Method of promoting remyelination

ABSTRACT

A method of promoting remyelination in a subject in need thereof includes administering to the subject a therapeutically effective amount of at least one (1,3) Diazole compound, wherein the therapeutically effective amount is the amount effective to induce endogenous oligodendrocyte precursor cell (OPC) differentiation in the subject&#39;s central nervous system.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.62/003,109, filed May 27, 2014 and is Continuation-in-Part ofInternational Application PCT/US2014/057471 filed Sep. 25, 2014, thesubject matter of which are incorporated herein by reference in theirentirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No.CON119651 awarded by the Myelin Repair Foundation. The United Statesgovernment has certain rights to the invention.

BACKGROUND

Multiple sclerosis (MS) is a complex neurological disease characterizedby deterioration of central nervous system (CNS) myelin. This insulatingmaterial, composed in its majority by lipids (70% lipids, 30% protein),protects axons and makes possible the saltatory conduction, which speedsaxonal electric impulse. Demyelination of axons in chronic MS may resultin axon degeneration and neuronal cell death, but more specifically, MSdestroys oligodendrocytes, the highly specialized CNS cells thatgenerate and maintain myelin.

Oligodendrocyte precursors (PDGFRα+, NG2-proteoglycan+), the immatureoligodendrocytes, are generated in ventral areas of the developing brainfrom a common glial progenitor, actively migrate and proliferatepopulating the CNS to finally differentiate to premyelinatingoligodendrocytes (O4+). At this maturation point, oligodendrocytes bothtarget and extend myelin sheaths along axons or they die. Less exploredhas been however, the hypothesis of remyelination by either endogenousoligodendrocyte precursors or transplanted cells.

Oligodendrocyte progenitor cells are abundant in demyelinated regions ofpatients with multiple sclerosis, yet fail to differentiate. Promotingremyelination by inducing differentiation and/or maturation ofendogenous oligodendrocyte progenitors can stimulate and enhanceintrinsic, natural remyelination thus reduction of clinical severity ofmyelination related disorders. Therefore, there is a need for compoundsand therapeutic methods capable inducing endogenous oligodendrocyteprecursor differentiation.

SUMMARY

Embodiments described herein generally relate to compounds and methodsfor promoting remyelination in a subject in need thereof as well as tomethods for the treatment of disease in subjects where myelinationand/or remyelination by the induction of endogenous oligodendrocyteprecursor differentiation is beneficial to the subject.

In some embodiments a method of promoting remyelination in a subject inneed thereof is provided. The method includes administering to thesubject a therapeutically effective amount of at least one (1,3) Diazolecompound, wherein the therapeutically effective amount is the amounteffective to induce endogenous oligodendrocyte precursor cell (OPC)differentiation in the subject's central nervous system. In someembodiments, the at least one (1,3) diazole compound or analog thereofhaving the formula (I):

where R₁ is a substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heteroaryl, heterocycloalkenyl containingfrom 5-6 ring atoms (wherein from 1-3 of the ring atoms is independentlyselected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), O, and S),C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, —Si(C₁-C₃ alkyl)₃, hydroxyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl,C₂-C₂₄ alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato,carbamoyl, C₁-C₂₄ alkyl-carbamoyl, arylcarbamoyl, thiocarbamoyl,carbamido, cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido,formyl, thioformyl, amino, C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄alkylamido, C₆-C₂₀ arylamido, imino, alkylimino, arylimino, nitro,nitroso, sulfo, sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄alkylsulfinyl, C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀arylsulfonyl, phosphono, phosphonato, phosphinato, phospho, phosphino,and combinations thereof, or pharmaceutically acceptable salts thereof.

Another embodiment relates to a method of treating a neurodegenerativedisease in a subject in need thereof. The method includes administeringto the subject a therapeutically effective amount of at least one (1,3)Diazole compound, wherein the therapeutically effective amount is theamount effective to induce endogenous oligodendrocyte precursor cell(OPC) differentiation and promote myelination in the subject's centralnervous system. In some embodiments, the at least one (1,3) diazolecompound or analog thereof having the formula (I):

where R₁ is a substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heteroaryl, heterocycloalkenyl containingfrom 5-6 ring atoms (wherein from 1-3 of the ring atoms is independentlyselected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), O, and S),C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, —Si(C₁-C₃ alkyl)₃, hydroxyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl,C₂-C₂₄ alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato,carbamoyl, C₁-C₂₄ alkyl-carbamoyl, arylcarbamoyl, thiocarbamoyl,carbamido, cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido,formyl, thioformyl, amino, C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄alkylamido, C₆-C₂₀ arylamido, imino, alkylimino, arylimino, nitro,nitroso, sulfo, sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄alkylsulfinyl, C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀arylsulfonyl, phosphono, phosphonato, phosphinato, phospho, phosphino,and combinations thereof, or pharmaceutically acceptable salts thereof.

Other embodiments relate to a method of treating a myelin relateddisorder in a subject in need thereof. The method includes administeringto the subject a therapeutically effective amount of at least one (1,3)Diazole compound, wherein the at least one (1,3) diazole compound oranalog thereof having the formula (I):

where R₁ is a substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heteroaryl, heterocycloalkenyl containingfrom 5-6 ring atoms (wherein from 1-3 of the ring atoms is independentlyselected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), O, and S),C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, —Si(C₁-C₃ alkyl)₃, hydroxyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl,C₂-C₂₄ alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato,carbamoyl, C₁-C₂₄ alkyl-carbamoyl, arylcarbamoyl, thiocarbamoyl,carbamido, cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido,formyl, thioformyl, amino, C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄alkylamido, C₆-C₂₀ arylamido, imino, alkylimino, arylimino, nitro,nitroso, sulfo, sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄alkylsulfinyl, C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀arylsulfonyl, phosphono, phosphonato, phosphinato, phospho, phosphino,and combinations thereof, or pharmaceutically acceptable salts thereof,and wherein the therapeutically effective amount is the amount effectiveto induce endogenous oligodendrocyte precursor cell (OPC)differentiation and promote myelination in the subject's central nervoussystem.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A-E) illustrate a pluripotent stem cell-based phenotypicscreening platform to identify modulators of OPC differentiation andmaturation. (A), Flow diagram depicting organization of primary screenas well as validation of putative drug hits. (B), RNAseq expression datashowing down regulation of pluripotent stem cell transcripts and upregulation of OPC transcripts when EpiSCs are differentiated into OPCs.Fragments per kilobase exon per million reads (FPKM) for each transcriptare shown as compared to in vivo isolated OPCs. (C), Representativeimages of vehicle and small molecule hit treated OPCs from the primaryscreen. Nuclear (DAPI) and myelin basic protein (MBP) staining alongwith high content analysis (HCA) to identify oligodendrocyte nuclei andMBP+ processes. Scale bar 100 μm. (D), Primary screen results displayedas normalized values of MBP process length and MBP process intensity forall 727 drugs. Baseline (vehicle) was set at 0 and thyroid hormone(positive control) was set at 100. The 21 drug hits were greater than 5standard deviations above baseline. (E), Chart ranking the 21 primarydrug hits into 4 tiers based on calculation of half maximal effectiveconcentration (EC50) and 50% toxicity (50% Tox) dose calculated from 7point dose treatment of OPCs.

FIGS. 2(A-D) illustrate four validated drugs promote precocious MBP+axonal ensheathment in ex vivo organotypic cerebellar slice cultures.(A), Flow diagram depicting organization of cerebellar slice assay andvalidation by western blot. (B), Example of MBP staining of a mouse P7cerebellar slice treated with drug for 5 days along with high contentanalysis (HCA) to identify aligned MBP+ fibers. Scale bar 100 μm. (C),Chart ranking 11 drugs from the primary screen into 3 groups (High,Medium, Low) based on their ability to increase MBP+ axonal ensheathmentrelative to vehicle (DMSO) treated controls as measured by HCA. n=6-12medial cerebellar slices per drug. Primary screen tier classificationfrom FIG. 1 are repeated for comparison; green (Tier A), (Tier B), and(Tier C). (D), Validation of HCA of drug treated cerebellar slices bywestern blot. Shown are fold changes in MBP levels for 4 drugs comparedto vehicle (DMSO) treated controls.

FIGS. 3(A-E) illustrate miconazole and clobetasol reduce diseaseseverity and promote remyelination in chronic MOG35-55 EAE mouse model.(A), Flow diagram depicting organization of EAE experimental design andanalysis of five drugs. A clinical scoring system where 1=limp tail;2=paralysis of one hind limb; and 3=paralysis of both hind limbs wasemployed. Mice were treated with drug or vehicle at peak of disease(˜day 15 after immunization; score of 3). (B), Three drugs showedsignificant reduction of EAE clinical scores after 10 days of treatmentin an initial treatment cohort (n=6 mice per group). (C) and (D). Thesethree drugs were repeated for further validation with a larger treatmentcohort (n=12 to 18 mice per group). The average clinical scores (C) andcumulative clinical scores (D) of miconazole and clobetasol treatedanimals showed significant reduction of disease and functionalimprovement. (E), Miconazole and clobetasol treated animals showedimprovements in white matter histology compared to vehicle treatedcontrols. Representative images of luxol fast blue (LFB) staining showeda clear decrease in areas of white matter disruption in the spinal cordsof drug treated animals which coincides with increased MBP staining.IBA1 staining showed a small reduction but not an abrogation of immunecell infiltration into the lesion areas. Representative toluidine bluestained images and electron micrographs revealed areas of significantremyelination in drug treated animals. Lesioned areas are outlined withdotted lines. Insets in toluidine blue staining show highermagnification of myelination in the corresponding spinal cords. *p<0.05,**p<0.01. Arrow in C, indicates beginning of drug treatment at diseasepeak and error bars are standard deviation of mean. (E) Scale bar 100μm, electron micrograph scale bar 2 μm. (B and C) Values are mean±SEM.(D) Dots each represent an individual animal with group mean (horizontalline), SD (box), and range (vertical line) depicted.

FIGS. 4(A-F) illustrate miconazole and clobetasol function throughpathways conserved in mouse OPC development and in human OPCs. (A),Overview of four orthogonal assays applied to clobetasol and miconazole.(B), MBP immunohistochemistry showing corpus callosum (CC) of P7 pupstreated for four days with drug or vehicle. Clobetasol and miconazoleshowed a significant increase in the length of the CC stained withaligned MBP+ fibers. *p<0.01, Striatum is marked as Str. (C),Representative phase contrast image of hESC colony cultured on matrigel.(D), Representative phase contrast image of hESC-derived OPCs. (E),hESC-derived OPCs stain positive for Sox10. (F), Representative imagesof hES-derived OPCs treated with vehicle (DMSO) or miconazole orclobetasol for 7 days. PLP staining is shown in green (top) along withhigh content analysis (HCA; bottom) to identify oligodendrocyte nucleiand PLP+ processes. (B, C, E, and F) Scale bars 100 μm. (D) Scale bar 50μm. (B) Values are mean±SEM.

FIGS. 5(A-C) illustrate in vitro phenotypic screen assay development.(A), Two epiblast stem cell (EpiSC)-derived oligodendrocyte progenitorcell (OPC) batches of greater than 100 million cells were generated andused for this study. These batches were derived from independent EpiSClines of distinct mouse strain and opposite gender. (B), The batches ofEpiSC-derived OPCs were sorted to purity (circled areas of FACS plots)using OPC cell surface markers NG2 and CD140α prior to use in thisstudy. (C), DMSO (v/v) tolerance of EpiSC derived-OPCs in 96-wellplates. (n=16 wells each condition; 16 20× fields sampled/well;mean±SEM). For reference, 0.05% (v/v) DMSO was used as vehicle for allin vitro experiments in this study.

FIGS. 6(A-D) illustrate performance of the primary screen of 727 hits on10 assay plates. (A), Thyroid hormone (positive control) and DMSOvehicle treatments yielded a consistent number of live cells imaged perwell across all 10 assay plates. Values are mean±SEM. (B), Signal tobackground (S/B) mean values with standard deviation (s.d.) of theentire screen were within an acceptable range to enable identificationof primary hits. (C), Raw data of myelin basic protein (MBP) processlength from the primary screen for thyroid hormone treatment and DMSOvehicle. (D), Raw data of MBP process intensity from the primary screenfor thyroid hormone treatment and DMSO vehicle. (C and D) Values aremean±SD. (A-D) 30,000 cells were seeded per well and approximately ⅓ ofeach well (n=8 wells each condition; 24 10× fields sampled/well) imagedyielding close to 10,000 cells sampled per well.

FIGS. 7(A-B) illustrate the dose response of miconazole. (A),Representative images of miconazole treated OPCs in dose response alongwith thyroid hormone (positive control) and vehicle (DMSO). Slighttoxicity and a decrease in PLP1 positive cells was observed only at thehighest 6.7 μM dose. Scale bar 100 μm. (B), Seven point dose responsecurve generated through script analysis of percent PLP1+ cells. Mean±SEMplotted.

FIGS. 8(A-B) illustrate the dose response of clobetasol. A,Representative images of clobetasol treated OPCs in dose response alongwith thyroid hormone (positive control) and vehicle (DMSO). Scale bar100 μm. B, Seven point dose response curve generated through scriptanalysis of percent PLP1+ cells. Mean±SEM plotted.

FIG. 9 illustrates high content imaging analysis of drug treatedcerebellar slices. Montaged images of postnatal day 7 mouse cerebellarslices treated with drug or vehicle for 5 days and stained for myelinbasic protein (MBP). Insets are a single field and inset analysisrepresents script identification and texture analysis of MBP positivealigned fibers. Representative MBP-stained areas called and included inscript analysis. Scale bar 100 μm.

FIG. 10 illustrates spinal cords from MOG35-55 immunized EAE animalsshow regions of repair and remyelination. Cervical spinal cords frommiconazole, clobetasol and benztropine treated animals show a decreasein areas of myelin disruption in the white matter as assayed by luxolfast blue and MBP immunohistochemistry. Lesions in the spinal cord aremarked by dotted lines. Scale bar 100 μm.

FIGS. 11(A-B) illustrate specificity of drug induced differentiation.(A), Representative images of OPCs treated with miconazole or clobetasolto evaluate drug induced astrocyte differentiation. Addition of both LIFand BMP served as a positive control. (B), Percent of astrocytesrelative to dose response 6.7, 5, 3.3, 1.7, 0.7, 0.5, 0.3 μM of eachdrug. Positive control (+) is LIF and BMP treatment of OPCs to induceastrocyte differentiation (n=4 wells each condition; 14 20× fieldssampled/well; mean±SEM). Scale bar 100 μm.

FIGS. 12(A-D) illustrate RNAseq on drug treated OPCs. (A), Volcano plotsof all genes from OPCs treated with clobetasol or miconazole relative tovehicle control, with differentially expressed genes highlighted (red).Significance (measured as −log 10[qvalue]) is plotted in relationship toexpression change (log 2[treatment/vehicle]). Time course was after 2, 6and 12 hours of drug treatment. (B), Venn diagrams depicting the overlapof genes differentially expressed at any time point and increased intreatments vs vehicle (left), as well as those decreased in treatmentsvs vehicle (right). (C), Canonical pathways perturbed by each drugtreatment according to Ingenuity Pathway Analysis. (D), Concurrent withRNAseq experiments, parallel drug treated cultures were allowed tomature into oligodendrocytes for 3 days in 12-well plates and analyzedto confirm drug efficacy in these experiments. n=3 wells each condition;45 10× fields sampled/well; mean±SEM.

FIG. 13 illustrates NIH Clinical Collection imidazole structure activityrelationship based on EAE validated hit miconazole. Primary screen rankout of 727 drugs (average of both length and intensity parameters), inparentheses. All drugs tested at 5 μM. Imidazole structure used forsearch [(1,3) diazole or (1,2,4)Triazole] highlighted.

FIG. 14 illustrates NIH Clinical Collection steroid structure activityrelationship based on EAE validated hit clobetasol. Primary screen rankout of 727 drugs (average of both length and intensity parameters), inparentheses. All drugs tested at 5 μM. Steroid structure used for searchis highlighted.

FIGS. 15(A-F) illustrate a pluripotent stem cell-based phenotypicscreening platform to identify modulators of OPC differentiation andmaturation. (A) Representative images of vehicle- and drug-hit-treatedmouse EpiSC-derived OPCs from the primary screen. Nuclear (DAPI(4′,6-diamidino-2-phenylindole), blue) and MBP (red) staining along withHCA to identify oligodendrocyte (oligo.) nuclei (green) and MBP+processes (yellow). Scale bar, 100 μm. (B) Scatter plot of primaryscreen results displayed as normalized values of MBP process length andintensity for all 727 drugs with the 22 hits marked in red. Baseline(vehicle) was set at zero and thyroid hormone (positive control) was setat 100. (C) Montaged images of whole postnatal day 7 mouse cerebellarslices treated with drug or vehicle for 5 days and stained for MBP(green). Insets show a representative example of the HCA script used toidentify and quantify MBP+-aligned fibres (light blue). Scale bars, 1 mmfor whole slices and 100 μm for insets. (D) Relative quantification ofHCA and western blot data from cerebellar slices treated for 5 days. ForHCA screen, n=1 with 6-12 slices averaged per group. For western blot,n=3 independent replicates of 12 slices per group. Values are mean forHCA and mean±s.e.m. for western blot. (E) Representative western blot ofMBP isoforms and f3-actin (loading control) of cerebellar slices treatedfor 5 days. (F) Chemical structures of clobetasol and miconazole.

FIGS. 16(A-D) illustrate miconazole and clobetasol each enhanceremyelination in the LPC lesion mouse model. (A) Representativeimmunohistochemical images of treated mice showing newly generatedoligodendrocytes (CC1, red) and MBP (green) within the lesion(approximated by white dashed outline) at eight and 12 d.p.l. Scale bar,200 μm. (B) Quantification of CC1⁺ oligodendrocytes per lesion area at 8d.p.l. Values are mean±s.e.m.; n=3 mice per group. Two-tailed t-test,*P<0.05. (C) Representative electron micrographs showing remyelinatedaxons within lesions of drug-treated mice at 12 d.p.l. Scale bar, 2 μm.(D) Scatter plot of g ratios of lesion axons at 12 d.p.l.; n=100calculated from two mice per group compared to wild-type intact axons.Percentage of lesion axons myelinated is indicated in the legend.

FIGS. 17(A-H) illustrate cellular and molecular effects of miconazoleand clobetasol on mouse OPCs. (A) Percentage MBP+ oligodendrocytesgenerated from OPCs at 72 h with treatments initiated at time pointsindicated; n=6 wells per condition with >6,000 cells scored per well.(B) Percentage MBP⁺ oligodendrocytes generated from OPCs treatedsimultaneously and analyzed at time points indicated; n=8 wells percondition with >1,700 cells scored per well. (C) Percentage GFAP⁺astrocytes generated from OPCs at 72 h of treatment; n=4 wells percondition >2,900 cells scored per well. (D) Heat map depictingbiochemical inhibition of muscarinic receptors M1-M5 displayed aspercentage inhibition with minimum (green) and maximum (red). (E)Western blot of total glucocorticoid receptor and its phosphorylation atSer220 (p-GR) in OPCs treated for 1 h. (F) Percentage MBP⁺oligodendrocytes generated from OPCs 72 h after treatment; n=6 wells percondition with >1,400 cells scored per well. (G) Western blot of totalERK1/2 and their phosphorylation at Thr202/Tyr204 or Thr185/Tyr187(p-ERK1/2) in cells (OPCs or mouse embryonic fibroblasts) treated for 1h. FGF served as a positive control for p-ERK1/2 induction. (H) Westernblot of total ERK1/2 and p-ERK1/2 in OPCs treated for 1 h in thepresence of the indicated pathway inhibitors. All graphs depictmean±s.e.m.

FIGS. 18(A-D) illustrate the therapeutic efficacy of miconazole andclobetasol in mouse models of MS. (A) Scoring of disease severity inrelapsing remitting PLP₁₃₉₋₁₅₁ induced EAE mice treated beginning on day13 (black arrow) and ending on day 29; n=10 mice per group. Graphdepicts mean daily disease score±s.e.m. (B) Flow-cytometric-basedquantification of spleen cell numbers at day 29 from the PLP₁₃₉₋₁₅₁ EAEcohort in A. Values are mean±s.e.m.; n=4 or 5 mice per group. (C)Scoring of disease severity in chronic progressive MOG₃₅₋₅₅-induced EAEmice treated daily for 10 days beginning at the peak of disease on day15 (black arrow); n=12-16 mice per group. Graph depicts mean dailydisease score±s.e.m. (D) Mean improvement in disease score per animal(peak score minus ending score) of MOG₃₅₋₅₅ EAE cohort in C. Also shownare external validation results in MOG₃₅₋₅₅ EAE from an independentcontract laboratory. n=12 mice per group. For all EAE experiments, drugswere dosed daily by intraperitoneal injection: clobetasol (2 mg/kg),miconazole (10 mg/kg), benztropine (10 mg/kg), or FTY720 (1 mg/kg). AllEAE disease scoring was as follows: 0, no abnormality; 1, limp tail; 2,limp tail and hind limb weakness; 3, hind limb paralysis; 4, hind limbparalysis and forelimb weakness; and 5, moribund. Two-tailed t-test,*P<0.05 and **P<0.01 for drug-treated groups compared with theirrespective vehicle-treated group.

FIGS. 19(A-G) illustrate the performance of the primary screen. (A)Representative flow cytometry plots showing co-expression of NG2 andCD140a in both batches of EpiSC-derived OPCs used for this study. Thebatches of EpiSC-derived OPCs were sorted to purity (circled areas ofplots) before use in this study. (B) RNaseq expression heat map showingdownregulation of pluripotent stem cell transcripts and upregulation ofOPC transcripts when EpiSCs were differentiated into OPCs. Fragments perkilobase exon per million reads (FPKM) for each transcript are showncompared with in vivo isolated mouse OPCs. (C) Quantification of DMSO(v/v) tolerance of EpiSC derived OPCs in 96-well plates shown asmean±s.e.m. For reference, 0.05% (v/v) DMSO was used as vehicle for allin vitro experiments in this study; n=16 wells per group with >690 cellsscored per well. (D) Quantification of cell viability of thyroid hormone(positive control) and DMSO vehicle treatments per well across all tenassay plates shown as mean±s.e.m.; n=80 wells per group with >6,800cells scored per well. (E) Signal to background (S/B) mean values withstandard deviation (s.d.) of controls from the entire screen; n=80 wellsper group. (F) Raw data of MBP process length from the primary screenfor thyroid hormone treatment and DMSO vehicle across all plates shownas mean±s.d.; n=8 wells per group with >6,800 cells scored per well. (G)Raw data of MBP process intensity from the primary screen for thyroidhormone treatment and DMSO vehicle shown as mean±s.d.; n=8 wells pergroup with >6,800 cells scored per well.

FIGS. 20(A-B) illustrate drag hit making and validation. (A) Chartranking the 22 primary drug hits (single dose rank) into four tiers onthe basis of calculation of EC₅₀ to induce PLP1⁺ oligodendrocytes fromOPCs and the concentration at which 50% of the cells were lost (50% Tox)calculated from a seven-point dose treatment; n=4 wells per dose perdrug using independently sourced drug and separate OPC batch from theprimary screen. Tiers ranged from the most potent and least toxiceffectors to the least potent and most toxic: tier a (green), tier b(grey), tier c (orange), and tier d (red). The 1,536-well formatexternal validation of 14 out of 16 tested hits is also shown. Drugswere further ranked into groups of high (green), medium (grey), and low(orange) on the basis of their ability to increase MBP axonalensheathment in mouse cerebellar brain slices relative to vehicle(DMSO)-treated controls as measured by HCA. NT, not tested. (B) Externalvalidation whole 1,536-well images of MBP (green) oligodendrocytesgenerated from OPCs after 72 h of treatment. GE InCell HCA is shown withprocesses traced in yellow and nuclei in blue.

FIGS. 21(A-D) illustrate primary screen structure-activity analysis.Chemoinformatic identification of two substructures consistentlyenriched in high-performing drugs in the OPC assay. Numerical activityrank in the primary screen is indicated with the top 22 shown in green,23-50 shown in grey, and 0.51 in red. (A) 1,3-Diazoles, mono-substitutedat the 1-position showed consistent activity on OPCs. b, c,1,3-Diazoles, poly-substituted at two or more of the R groups (B) or1,2,4-triazoles, mono-substituted at the 1-position (C) showed noactivity on OPCs. (D) The sterane base structure showed enrichment inthe top performing hits.

FIGS. 22(A-B) illustrate histological assessment of remyelination in theLPC-induced model of demyelination. (A) Representative electronmicrographs showing remyelinated axons within lesions of miconazoletreated mice at 8 d.p.l. Scale bar, 2 mm. (B) Histological sectionsstained with toluidine blue showing the extent of remyelination in thelesions of treated animals at 12 d.p.l. Normal uninjured myelin appearsto the left of the black dashed line demarcating the definitive lesionedge. Scale bar, 20 mm.

FIGS. 23(A-B) Miconazole and clobetasol enhance myelination in vivo. (A)(B) Representative immunohistochemical images of the lateral corpuscallosum (CC) of postnatal day 6 mouse pups that had been injectedintraperitoneally daily for 4 days previously starting on postnatal day2 with vehicle, clobetasol (2 mg/kg), or miconazole (10 mg/kg). CC1(red) marks newly generated oligodendrocytes (a) and MBP (green) showsthe extent of developmental myelination (b). Clobetasol and miconazoletreatment each induce a marked increase in the number of CC1-positiveoligodendrocytes in the lateral corpus callosum (A) and a significantincrease in the length of the corpus callosum covered with aligned MBPfibres (B). Scale bar, 200 mm. Twotailed t-test, *P≤0.05 and **P≤0.01.Str, striatum. All graphs are presented as mean±s.e.m.

FIGS. 24(A-C) illustrate RNaseq time course of drug-treated OPCs. (A)Volcano plots of all genes from OPCs treated with clobetasol ormiconazole relative to vehicle control, with differentially expressedgenes highlighted (red). Significance (measured as −log₁₀ [q value]) isplotted in relation to expression change (log₂ [treatment/vehicle]).Time course was after 2, 6, and 12 h of drug treatment. (B) Venndiagrams depicting the overlap of genes differentially expressed at anytime point and increased in treatments versus vehicle (left), as well asthose decreased in treatments versus vehicle (right). (C) Significantcanonical pathways perturbed by each drug treatment according toIngenuity Pathway Analysis.

FIGS. 25(A-C) illustrate Global phosphoproteomic analysis ofmiconazole-treated OPCs. (A, B) OPCs treated with miconazole for (A) 1 hor (B) 5 h followed by global phosphoproteomic analysis. Proteinshighlighted in green were observed to have a twofold or greater increasein phosphorylation whereas those highlighted in red were observed tohave a twofold or greater decrease in phosphorylation compared withtime-point-matched vehicle treated controls. Proteins highlighted ingrey were detected in the analysis but were not changed compared withvehicle control. (C) Quantification of the percentage of MBPoligodendrocytes differentiated from mouse OPCs after 72 h of treatmentwith DMSO, miconazole (1 μM), or voriconazole (seven doses, 0.01-6.7μM); n=6 wells per condition with 6,000 cells scored per well. Graphpresented as mean±s.e.m. The chemical structure of voriconazole isshown.

FIGS. 26(A-G) illustrate miconazole and clobetasol enhance human OPCdifferentiation. (A) Representative phase contrast image of a hESCcolony cultured on matrigel. (B), Representative phase contrast image ofhESC-derived OPCs. (C) hESC-derived OPCs stain positive for Sox10. (D,E)Representative images of hESC-derived OPCs (d) and hiPSC-derived OPCs(e) treated with vehicle (DMSO), miconazole (1 μM), or clobetasol (5 μM)for 21 days stained for MBP (red). (F,G) HCA of hESC-derived (F) andhiPSC-derived (G) OPCs differentiated in the presence of drugs orvehicle over 21 days; n=3-5 wells per condition with >120 cells scoredper well. Graphs presented as mean±s.e.m. Scale bars, 100 mm.

FIGS. 27(A-J) illustrate the effects of miconazole and clobetasol onimmune cell survival and function. (A-D) Quantification of cellproliferation (A, C) and differentiation (B, D) of naive CD4⁺ T cellsfrom unprimed SJL/J mice after activation with plate-bound anti-CD3under Th1 (A, B) or Th17 (C, D) cell driving conditions. (E-J) Ex vivorecall assays quantifying cell proliferation (DCPM) (E, H), with IFN-γ(F, I) and IL-17 (G, J) cytokine production from lymphocytes of miceprimed with PLP₁₃₉₋₁₅₁ (E-G) or MOG₃₅₋₅₅ (H-J). Cultures were treatedwith vehicle (DMSO), benztropine, clobetasol, or miconazole (10⁻⁹-10⁻⁵M) and analysed after 4 days. Four independent replicates are shown foreach assay.

FIGS. 28(A-E) illustrate Histological improvements in MOG35-55 EAEspinal cords after treatment with miconazole or clobetasol. (A), (B)Representative images of luxol fast blue (LFB) staining (A) demonstrateda clear decrease in areas of white matter disruption in the spinal cordsof drug treated animals which coincides with increased MBP staining (B).(C) IBA1 staining showed a small reduction of immune cell infiltrationinto the lesion areas, especially in clobetasol-treated animals, but notan abrogation. (D, E) Representative images stained with toluidine blue(D) and electron micrographs (E) revealed a reduction in the areas ofdemyelination in drug treated animals. Lesioned areas are outlined withblack dotted lines. Insets in toluidine blue staining show highermagnification of myelination in the corresponding spinal cords. Scalebars, 100 mm (A-C, D) and 2 μm (E).

DETAILED DESCRIPTION

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises, such as Current Protocolsin Molecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates). Unlessotherwise defined, all technical terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which theapplication pertains. Commonly understood definitions of molecularbiology terms can be found in, for example, Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th Ed., Springer-Verlag: New York,1991, and Lewin, Genes V, Oxford University Press: New York, 1994. Thedefinitions provided herein are to facilitate understanding of certainterms used frequently herein and are not meant to limit the scope of theapplication.

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisapplication belongs.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “comprise,” “comprising,” “include,” “including,” “have,” and“having” are used in the inclusive, open sense, meaning that additionalelements may be included. The terms “such as”, “e.g.”, as used hereinare non-limiting and are for illustrative purposes only. “Including” and“including but not limited to” are used interchangeably.

The term “or” as used herein should be understood to mean “and/or”,unless the context clearly indicates otherwise.

As used herein, the term “about” or “approximately” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length. In oneembodiment, the term “about” or “approximately” refers a range ofquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%,±2%, or ±1% about a reference quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length.

It will be noted that the structure of some of the compounds of theapplication include asymmetric (chiral) carbon or sulfur atoms. It is tobe understood accordingly that the isomers arising from such asymmetryare included herein, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis. The compounds of thisapplication may exist in stereoisomeric form, therefore can be producedas individual stereoisomers or as mixtures.

The term “isomerism” means compounds that have identical molecularformulae but that differ in the nature or the sequence of bonding oftheir atoms or in the arrangement of their atoms in space. Isomers thatdiffer in the arrangement of their atoms in space are termed“stereoisomers”. Stereoisomers that are not mirror images of one anotherare termed “diastereoisomers”, and stereoisomers that arenon-superimposable mirror images are termed “enantiomers”, or sometimesoptical isomers. A carbon atom bonded to four nonidentical substituentsis termed a “chiral center” whereas a sulfur bound to three or fourdifferent substitutents, e.g. sulfoxides or sulfinimides, is likewisetermed a “chiral center”.

The term “chiral isomer” means a compound with at least one chiralcenter. It has two enantiomeric forms of opposite chirality and mayexist either as an individual enantiomer or as a mixture of enantiomers.A mixture containing equal amounts of individual enantiomeric forms ofopposite chirality is termed a “racemic mixture”. A compound that hasmore than one chiral center has 2n−1 enantiomeric pairs, where n is thenumber of chiral centers. Compounds with more than one chiral center mayexist as either an individual diastereomer or as a mixture ofdiastereomers, termed a “diastereomeric mixture”. When one chiral centeris present, a stereoisomer may be characterized by the absoluteconfiguration (R or S) of that chiral center. Alternatively, when one ormore chiral centers are present, a stereoisomer may be characterized as(+) or (−). Absolute configuration refers to the arrangement in space ofthe substituents attached to the chiral center. The substituentsattached to the chiral center under consideration are ranked inaccordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn etal, Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al.,Angew. Chem. 1966, 78, 413; Cahn and Ingold, J Chem. Soc. 1951 (London),612; Cahn et al., Experientia 1956, 12, 81; Cahn, J., Chem. Educ. 1964,41, 116).

The term “geometric Isomers” means the diastereomers that owe theirexistence to hindered rotation about double bonds. These configurationsare differentiated in their names by the prefixes cis and trans, or Zand E, which indicate that the groups are on the same or opposite sideof the double bond in the molecule according to the Cahn-Ingold-Prelogrules. Further, the structures and other compounds discussed in thisapplication include all atropic isomers thereof.

The term “atropic isomers” are a type of stereoisomer in which the atomsof two isomers are arranged differently in space. Atropic isomers owetheir existence to a restricted rotation caused by hindrance of rotationof large groups about a central bond. Such atropic isomers typicallyexist as a mixture, however as a result of recent advances inchromatography techniques, it has been possible to separate mixtures oftwo atropic isomers in select cases.

The terms “crystal polymorphs” or “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or salt or solvate thereof) cancrystallize in different crystal packing arrangements, all of which havethe same elemental composition. Different crystal forms usually havedifferent X-ray diffraction patterns, infrared spectral, melting points,density hardness, crystal shape, optical and electrical properties,stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

The term “derivative” refers to compounds that have a common corestructure, and are substituted with various groups as described herein.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres includeacyl sulfonimides, tetrazoles, sulfonates, and phosphonates. See, e.g.,Patani and LaVoie, Chem. Rev. 96, 3147-3176 (1996).

The phrases “parenteral administration” and “administered parenterally”are art-recognized terms, and include modes of administration other thanenteral and topical administration, such as injections, and include,without limitation, intravenous, intramuscular, intrapleural,intravascular, intrapericardial, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

The term “treating” is art-recognized and includes inhibiting a disease,disorder or condition in a subject, e.g., impeding its progress; andrelieving the disease, disorder or condition, e.g., causing regressionof the disease, disorder and/or condition. Treating the disease orcondition includes ameliorating at least one symptom of the particulardisease or condition, even if the underlying pathophysiology is notaffected.

The term “preventing” is art-recognized and includes stopping a disease,disorder or condition from occurring in a subject, which may bepredisposed to the disease, disorder and/or condition but has not yetbeen diagnosed as having it. Preventing a condition related to a diseaseincludes stopping the condition from occurring after the disease hasbeen diagnosed but before the condition has been diagnosed.

The term “pharmaceutical composition” refers to a formulation containingthe disclosed compounds in a form suitable for administration to asubject. In a preferred embodiment, the pharmaceutical composition is inbulk or in unit dosage form. The unit dosage form is any of a variety offorms, including, for example, a capsule, an IV bag, a tablet, a singlepump on an aerosol inhaler, or a vial. The quantity of active ingredient(e.g., a formulation of the disclosed compound or salts thereof) in aunit dose of composition is an effective amount and is varied accordingto the particular treatment involved. One skilled in the art willappreciate that it is sometimes necessary to make routine variations tothe dosage depending on the age and condition of the patient. The dosagewill also depend on the route of administration. A variety of routes arecontemplated, including oral, pulmonary, rectal, parenteral,transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal,intranasal, inhalational, and the like. Dosage forms for the topical ortransdermal administration of a compound described herein includespowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, nebulized compounds, and inhalants. In a preferred embodiment,the active compound is mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants that are required.

The term “flash dose” refers to compound formulations that are rapidlydispersing dosage forms.

The term “immediate release” is defined as a release of compound from adosage form in a relatively brief period of time, generally up to about60 minutes. The term “modified release” is defined to include delayedrelease, extended release, and pulsed release. The term “pulsed release”is defined as a series of releases of drug from a dosage form. The term“sustained release” or “extended release” is defined as continuousrelease of a compound from a dosage form over a prolonged period.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, polymers and othermaterials and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” is art-recognized, andincludes, for example, pharmaceutically acceptable materials,compositions or vehicles, such as a liquid OR solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting any subject composition from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof a subject composition and not injurious to the patient. In certainembodiments, a pharmaceutically acceptable carrier is non-pyrogenic.Some examples of materials which may serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

The compounds of the application are capable of further forming salts.All of these forms are also contemplated herein.

“Pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. For example, the saltcan be an acid addition salt. One embodiment of an acid addition salt isa hydrochloride salt. The pharmaceutically acceptable salts can besynthesized from a parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrilebeing preferred. Lists of salts are found in Remington's PharmaceuticalSciences, 18th ed. (Mack Publishing Company, 1990).

The compounds described herein can also be prepared as esters, forexample pharmaceutically acceptable esters. For example, a carboxylicacid function group in a compound can be converted to its correspondingester, e.g., a methyl, ethyl, or other ester. Also, an alcohol group ina compound can be converted to its corresponding ester, e.g., anacetate, propionate, or other ester.

The compounds described herein can also be prepared as prodrugs, forexample pharmaceutically acceptable prodrugs. The terms “pro-drug” and“prodrug” are used interchangeably herein and refer to any compound,which releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.) the compounds can bedelivered in prodrug form. Thus, the compounds described herein areintended to cover prodrugs of the presently claimed compounds, methodsof delivering the same and compositions containing the same. “Prodrugs”are intended to include any covalently bonded carriers that release anactive parent drug in vivo when such prodrug is administered to asubject. Prodrugs are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds wherein a hydroxy, amino, sulfhydryl, carboxy, orcarbonyl group is bonded to any group that may be cleaved in vivo toform a free hydroxyl, free amino, free sulfhydryl, free carboxy or freecarbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates, andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxy functional groups, ester groups (e.g., ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases andenaminones of amino functional groups, oximes, acetals, ketals and enolesters of ketone and aldehyde functional groups in compounds of FormulaI, and the like, See Bundegaard, H. “Design of Prodrugs” p 1-92,Elesevier, New York-Oxford (1985).

The term “protecting group” refers to a grouping of atoms that whenattached to a reactive group in a molecule masks, reduces or preventsthat reactivity. Examples of protecting groups can be found in Green andWuts, Protective Groups in Organic Chemistry, (Wiley, 2.sup.nd ed.1991); Harrison and Harrison et al., Compendium of Synthetic OrganicMethods, Vols. 1-8 (John Wiley and Sons, 1971-1996); and Kocienski,Protecting Groups, (Verlag, 3^(rd) ed. 2003).

The term “amine protecting group” is intended to mean a functional groupthat converts an amine, amide, or other nitrogen-containing moiety intoa different chemical group that is substantially inert to the conditionsof a particular chemical reaction. Amine protecting groups arepreferably removed easily and selectively in good yield under conditionsthat do not affect other functional groups of the molecule. Examples ofamine protecting groups include, but are not limited to, formyl, acetyl,benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, t-butyloxycarbonyl(Boc), p-methoxybenzyl, methoxymethyl, tosyl, trifluoroacetyl,trimethylsilyl (TMS), fluorenyl-methyloxycarbonyl,2-trimethylsilyl-ethyoxycarbonyl, 1-methyl-1-(4-biphenylyl)ethoxycarbonyl, allyloxycarbonyl, benzyloxycarbonyl (CBZ),2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted tritylgroups, 9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl(NVOC), and the like. Those of skill in the art can identify othersuitable amine protecting groups.

Representative hydroxy protecting groups include those where the hydroxygroup is either acylated or alkylated such as benzyl, and trityl ethersas well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethersand allyl ethers.

Additionally, the salts of the compounds described herein, can exist ineither hydrated or unhydrated (the anhydrous) form or as solvates withother solvent molecules. Nonlimiting examples of hydrates includemonohydrates, dihydrates, etc. Nonlimiting examples of solvates includeethanol solvates, acetone solvates, etc.

The term “solvates” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

The compounds, salts and prodrugs described herein can exist in severaltautomeric forms, including the enol and imine form, and the keto andenamine form and geometric isomers and mixtures thereof. Tautomers existas mixtures of a tautomeric set in solution. In solid form, usually onetautomer predominates. Even though one tautomer may be described, thepresent application includes all tautomers of the present compounds. Atautomer is one of two or more structural isomers that exist inequilibrium and are readily converted from one isomeric form to another.This reaction results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Insolutions where tautomerization is possible, a chemical equilibrium ofthe tautomers will be reached. The exact ratio of the tautomers dependson several factors, including temperature, solvent, and pH. The conceptof tautomers that are interconvertable by tautomerizations is calledtautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs.

Tautomerizations can be catalyzed by: Base: 1. deprotonation; 2.formation of a delocalized anion (e.g., an enolate); 3. protonation at adifferent position of the anion; Acid: 1. protonation; 2. formation of adelocalized cation; 3. deprotonation at a different position adjacent tothe cation.

The term “analog” refers to a chemical compound that is structurallysimilar to another but differs slightly in composition (as in thereplacement of one atom by an atom of a different element or in thepresence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

A “patient,” “subject,” or “host” to be treated by the subject methodmay mean either a human or non-human animal, such as a mammal, a fish, abird, a reptile, or an amphibian. Thus, the subject of the hereindisclosed methods can be a human, non-human primate, horse, pig, rabbit,dog, sheep, goat, cow, cat, guinea pig or rodent. The term does notdenote a particular age or sex. Thus, adult and newborn subjects, aswell as fetuses, whether male or female, are intended to be covered. Inone aspect, the subject is a mammal. A patient refers to a subjectafflicted with a disease or disorder. In a particular aspect, thesubject is afflicted with a neurodegenerative disease or disorder. Incertain aspects, the subject is afflicted with a myelination relateddisorder.

The terms “prophylactic” or “therapeutic” treatment is art-recognizedand includes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, i.e., it protects thehost against developing the unwanted condition, whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The terms “therapeutic agent”, “drug”, “medicament” and “bioactivesubstance” are art-recognized and include molecules and other agentsthat are biologically, physiologically, or pharmacologically activesubstances that act locally or systemically in a patient or subject totreat a disease or condition. The terms include without limitationpharmaceutically acceptable salts thereof and prodrugs. Such agents maybe acidic, basic, or salts; they may be neutral molecules, polarmolecules, or molecular complexes capable of hydrogen bonding; they maybe prodrugs in the form of ethers, esters, amides and the like that arebiologically activated when administered into a patient or subject.

The phrase “therapeutically effective amount” or “pharmaceuticallyeffective amount” is an art-recognized term. In certain embodiments, theterm refers to an amount of a therapeutic agent that produces somedesired effect at a reasonable benefit/risk ratio applicable to anymedical treatment. In certain embodiments, the term refers to thatamount necessary or sufficient to eliminate, reduce or maintain a targetof a particular therapeutic regimen. The effective amount may varydepending on such factors as the disease or condition being treated, theparticular targeted constructs being administered, the size of thesubject or the severity of the disease or condition. One of ordinaryskill in the art may empirically determine the effective amount of aparticular compound without necessitating undue experimentation. Incertain embodiments, a therapeutically effective amount of a therapeuticagent for in vivo use will likely depend on a number of factors,including: the rate of release of an agent from a polymer matrix, whichwill depend in part on the chemical and physical characteristics of thepolymer; the identity of the agent; the mode and method ofadministration; and any other materials incorporated in the polymermatrix in addition to the agent. In certain embodiments, atherapeutically effective amount is the amount effective to induceendogenous oligodendrocyte precursor cell differentiation and/ormaturation, thereby promoting myelination in the subject's centralnervous system.

The term “ED50” is art-recognized. In certain embodiments, ED50 meansthe dose of a drug, which produces 50% of its maximum response oreffect, or alternatively, the dose, which produces a pre-determinedresponse in 50% of test subjects or preparations. The term “LD50” isart-recognized. In certain embodiments, LD50 means the dose of a drug,which is lethal in 50% of test subjects. The term “therapeutic index” isan art-recognized term, which refers to the therapeutic index of a drug,defined as LD50/ED50.

The terms “IC₅₀,” or “half maximal inhibitory concentration” is intendedto refer to the concentration of a substance (e.g., a compound or adrug) that is required for 50% inhibition of a biological process, orcomponent of a process, including a protein, subunit, organelle,ribonucleoprotein, etc.

With respect to any chemical compounds, the present application isintended to include all isotopes of atoms occurring in the presentcompounds. Isotopes include those atoms having the same atomic numberbut different mass numbers. By way of general example and withoutlimitation, isotopes of hydrogen include tritium and deuterium, andisotopes of carbon include C-13 and C-14.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent can be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent can be bonded via any atom in suchsubstituent. Combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

When an atom or a chemical moiety is followed by a subscripted numericrange (e.g., C₁₋₆), it is meant to encompass each number within therange as well as all intermediate ranges. For example, “C₁₋₆ alkyl” ismeant to include alkyl groups with 1, 2, 3, 4, 5, 6, 1-6, 1-5, 1-4, 1-3,1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, and 5-6 carbons.

The term “alkyl” is intended to include both branched (e.g., isopropyl,tert-butyl, isobutyl), straight-chain e.g., methyl, ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl), and cycloalkyl(e.g., alicyclic) groups (e.g., cyclopropyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. Such aliphatic hydrocarbon groupshave a specified number of carbon atoms. For example, C₁₋₆ alkyl isintended to include C₁, C₂, C₃, C₄, C₅, and C₆ alkyl groups. As usedherein, “lower alkyl” refers to alkyl groups having from 1 to 6 carbonatoms in the backbone of the carbon chain. “Alkyl” further includesalkyl groups that have oxygen, nitrogen, sulfur or phosphorous atomsreplacing one or more hydrocarbon backbone carbon atoms. In certainembodiments, a straight chain or branched chain alkyl has six or fewercarbon atoms in its backbone (e.g., C₁-C₆ for straight chain, C₃-C₆ forbranched chain), for example four or fewer. Likewise, certaincycloalkyls have from three to eight carbon atoms in their ringstructure, such as five or six carbons in the ring structure.

The term “substituted alkyls” refers to alkyl moieties havingsubstituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkyl,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “aralkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). If not otherwise indicated, the terms “alkyl” and “loweralkyl” include linear, branched, cyclic, unsubstituted, substituted,and/or heteroatom-containing alkyl or lower alkyl, respectively.

The term “alkenyl” refers to a linear, branched or cyclic hydrocarbongroup of 2 to about 24 carbon atoms containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl,cyclopentenyl, cyclohexenyl, cyclooctenyl, and the like. Generally,although again not necessarily, alkenyl groups can contain 2 to about 18carbon atoms, and more particularly 2 to 12 carbon atoms. The term“lower alkenyl” refers to an alkenyl group of 2 to 6 carbon atoms, andthe specific term “cycloalkenyl” intends a cyclic alkenyl group,preferably having 5 to 8 carbon atoms. The term “substituted alkenyl”refers to alkenyl substituted with one or more substituent groups, andthe terms “heteroatom-containing alkenyl” and “heteroalkenyl” refer toalkenyl or heterocycloalkenyl (e.g., heterocylcohexenyl) in which atleast one carbon atom is replaced with a heteroatom. If not otherwiseindicated, the terms “alkenyl” and “lower alkenyl” include linear,branched, cyclic, unsubstituted, substituted, and/orheteroatom-containing alkenyl and lower alkenyl, respectively.

The term “alkynyl” refers to a linear or branched hydrocarbon group of 2to 24 carbon atoms containing at least one triple bond, such as ethynyl,n-propynyl, and the like. Generally, although again not necessarily,alkynyl groups can contain 2 to about 18 carbon atoms, and moreparticularly can contain 2 to 12 carbon atoms. The term “lower alkynyl”intends an alkynyl group of 2 to 6 carbon atoms. The term “substitutedalkynyl” refers to alkynyl substituted with one or more substituentgroups, and the terms “heteroatom-containing alkynyl” and“heteroalkynyl” refer to alkynyl in which at least one carbon atom isreplaced with a heteroatom. If not otherwise indicated, the terms“alkynyl” and “lower alkynyl” include linear, branched, unsubstituted,substituted, and/or heteroatom-containing alkynyl and lower alkynyl,respectively.

The terms “alkyl”, “alkenyl”, and “alkynyl” are intended to includemoieties which are diradicals, i.e., having two points of attachment. Anonlimiting example of such an alkyl moiety that is a diradical is—CH₂CH₂—, i.e., a C₂ alkyl group that is covalently bonded via eachterminal carbon atom to the remainder of the molecule.

The term “alkoxy” refers to an alkyl group bound through a single,terminal ether linkage; that is, an “alkoxy” group may be represented as—O-alkyl where alkyl is as defined above. A “lower alkoxy” group intendsan alkoxy group containing 1 to 6 carbon atoms, and includes, forexample, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc.Preferred substituents identified as “C₁-C₆ alkoxy” or “lower alkoxy”herein contain 1 to 3 carbon atoms, and particularly preferred suchsubstituents contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).

The term “aryl” refers to an aromatic substituent containing a singlearomatic ring or multiple aromatic rings that are fused together,directly linked, or indirectly linked (such that the different aromaticrings are bound to a common group such as a methylene or ethylenemoiety). Aryl groups can contain 5 to 20 carbon atoms, and particularlypreferred aryl groups can contain 5 to 14 carbon atoms. Examples of arylgroups include benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles”, “heterocycles,” “heteroaryls” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diaryl amino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings, which are not aromatic so as to form amulticyclic system (e.g., tetralin, methylenedioxyphenyl). If nototherwise indicated, the term “aryl” includes unsubstituted,substituted, and/or heteroatom-containing aromatic substituents.

The term “alkaryl” refers to an aryl group with an alkyl substituent,and the term “aralkyl” refers to an alkyl group with an arylsubstituent, wherein “aryl” and “alkyl” are as defined above. Exemplaryaralkyl groups contain 6 to 24 carbon atoms, and particularly preferredaralkyl groups contain 6 to 16 carbon atoms. Examples of aralkyl groupsinclude, without limitation, benzyl, 2-phenyl-ethyl, 3-phenyl-propyl,4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl,4-phenylcyclohexylmethyl, 4-benzylcyclohexylmethyl, and the like.Alkaryl groups include, for example, p-methylphenyl, 2,4-dimethylphenyl,p-cyclohexylphenyl, 2,7-dimethylnaphthyl, 7-cyclooctylnaphthyl,3-ethyl-cyclopenta-1,4-diene, and the like.

The terms “heterocyclyl” or “heterocyclic group” include closed ringstructures, e.g., 3- to 10-, or 4- to 7-membered rings, which includeone or more heteroatoms. “Heteroatom” includes atoms of any elementother than carbon or hydrogen. Examples of heteroatoms include nitrogen,oxygen, sulfur and phosphorus.

Heterocyclyl groups can be saturated or unsaturated and includepyrrolidine, oxolane, thiolane, piperidine, piperazine, morpholine,lactones, lactams, such as azetidinones and pyrrolidinones, sultams, andsultones. Heterocyclic groups such as pyrrole and furan can havearomatic character. They include fused ring structures, such asquinoline and isoquinoline. Other examples of heterocyclic groupsinclude pyridine and purine. The heterocyclic ring can be substituted atone or more positions with such substituents as described above, as forexample, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,cyano, azido, heterocyclyl, or an aromatic or heteroaromatic moiety.Heterocyclic groups can also be substituted at one or more constituentatoms with, for example, a lower alkyl, a lower alkenyl, a lower alkoxy,a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, ahydroxyl, —CF₃, or —CN, or the like.

The term “halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.“Counterion” is used to represent a small, negatively charged speciessuch as fluoride, chloride, bromide, iodide, hydroxide, acetate, andsulfate.

The terms “substituted” as in “substituted alkyl,” “substituted aryl,”and the like, as alluded to in some of the aforementioned definitions,is meant that in the alkyl, aryl, or other moiety, at least one hydrogenatom bound to a carbon (or other) atom is replaced with one or morenon-hydrogen substituents. Examples of such substituents include,without limitation: functional groups such as halo, hydroxyl, silyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl(—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl),carboxy (—COOH), carboxylato (—COO—), carbamoyl (—(CO)—NH₂),mono-(C₁-C₂₄ alkyl)-substituted carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)),di-(C₁-C₄ alkyl)-substituted carbamoyl (—(CO)—N(C₁-C₂₄ alkyl)₂),mono-substituted arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl(—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano (—CN), isocyano (—N⁺C⁻),cyanato (—O—CN), isocyanato (—ON⁺C⁻), isothiocyanato (—S—CN), azido(—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino (—NH₂), mono-and di-(C₁-C₂₄ alkyl)-substituted amino, mono- and di-(C₅-C₂₀aryl)-substituted amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀arylamido (—NH—(CO)-aryl), imino (—CR═NH where R=hydrogen, C₁-C₂₄ alkyl,C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), alkylimino(—CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino(—CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro(—NO₂), nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl(—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl),C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl),C₅-C₂₀ arylsulfonyl (—SO₂-aryl), phosphono (—P(O)(OH)₂), phosphonato(—P(O)(O)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), and phosphino(—PH₂); and the hydrocarbyl moieties C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, and C₆-C₂₄ aralkyl.

In addition, the aforementioned functional groups may, if a particulargroup permits, be further substituted with one or more additionalfunctional groups or with one or more hydrocarbyl moieties such as thosespecifically enumerated above. Analogously, the above-mentionedhydrocarbyl moieties may be further substituted with one or morefunctional groups or additional hydrocarbyl moieties such as thosespecifically enumerated.

When the term “substituted” appears prior to a list of possiblesubstituted groups, it is intended that the term apply to every memberof that group. For example, the phrase “substituted alkyl, alkenyl, andaryl” is to be interpreted as “substituted alkyl, substituted alkenyl,and substituted aryl.” Analogously, when the term“heteroatom-containing” appears prior to a list of possibleheteroatom-containing groups, it is intended that the term apply toevery member of that group. For example, the phrase“heteroatom-containing alkyl, alkenyl, and aryl” is to be interpreted as“heteroatom-containing alkyl, substituted alkenyl, and substituted aryl.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, the phrase “optionally substituted” means that anon-hydrogen substituent may or may not be present on a given atom, and,thus, the description includes structures wherein a non-hydrogensubstituent is present and structures wherein a non-hydrogen substituentis not present.

The terms “stable compound” and “stable structure” are meant to indicatea compound that is sufficiently robust to survive isolation, and asappropriate, purification from a reaction mixture, and formulation intoan efficacious therapeutic agent.

The terms “free compound” is used herein to describe a compound in theunbound state.

Throughout the description, where compositions are described as having,including, or comprising, specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the compositionsand methods described herein remains operable. Moreover, two or moresteps or actions can be conducted simultaneously.

The term “small molecule” is an art-recognized term. In certainembodiments, this term refers to a molecule, which has a molecularweight of less than about 2000 amu, or less than about 1000 amu, andeven less than about 500 amu.

All percentages and ratios used herein, unless otherwise indicated, areby weight.

Embodiments described herein relate to methods of promotingremyelination in a subject in need thereof as well as treating amyelination related disorder in a subject in need thereof. Compoundsdescribed herein can be used in the treatment of myelination relatedneurodegenerative disorders, such as multiple sclerosis, to induce andpromote differentiation and/or maturation of endogenous oligodendrocyteprecursor cells, thereby promoting myelination or remyelination in somecases in the subject's central nervous system. The term “remyelination”as used herein refers to the process of creating new myelin sheaths ondemyelinated axons in the CNS.

The term “oligodendrocyte precursor cells” as used herein refersimmature or induced oligodendrocyte cells. Oligodendrocyte precursorcells can be identified by the expression of a number of surfaceantigens. For example, the surface antigens known as platelet-derivedgrowth factor-alpha receptor subunit (PDGFRα), NG2 chondroitin sulfateproteoglycan, and ganglioside GD3, are commonly used to identifyoligodendrocyte precursor cells.

Endogenous immature oligodendrocyte precursors are generated in ventralareas of the developing brain from a common glial progenitor. Theimmature cells actively migrate and proliferate populating the CNS tofinally differentiate to premyelinating oligodendrocytes (O4+).Oligodendrocyte precursor differentiation and maturation ischaracterized by an extension of multiple processes, increase in cellbody size and formation of myelin.

In some embodiments, the compounds for use in the methods describedherein are identified using a high-throughput small molecule screen thatis biased to identify compounds that have both a high potency and lowtoxicity in mammal subjects and are able to induce and promotedifferentiation and/or maturation of oligodendrocyte precursor cells orthat are capable of promoting remyelination in a subject in needthereof. The term “small molecule” as used herein refers to biologicallyactive organic compounds of low molecular weight (e.g. <500 kDa) whichmay cross biological membranes and modulate intracellular processes.

Briefly, the high-throughput small molecule screen can include a primaryscreening where small drug-like organic compounds (250-550 kDa) areadded to cells seeded on a multiwall plate (e.g., 96-well plate) andincubated. The cells are then visually screened for oligodendrocyteprecursor morphology changes. In a secondary screening, differentiationand maturation induced by selected compounds was further validated byfluorescence microscopy. Further oligodendrocyte precursordifferentiation and maturation in response to selected compounds can beassessed by induction of myelin protein expression as determined byimmunocytochemistry and western blot. Examples of assays that can beused in the primary and secondary screening are described in Najm et al.Nat Methods. 2011 Sep. 25; 8(11):957-62; Bai et al. Neurosci Bull. 2013April; 29(2):239-50; Yang et al. Dev Biol. 2011 Feb. 1; 350(1):127-38;and Cho et al. Curr Neuropharmacol. 2007 March; 5(1): 19-33

In some embodiments, the compounds can be further screened using a brainslice ex vivo assay that assesses myelination the brains of mammals,(e.g., rats and mice). Such assays are described, for example, in Bai etal. Neurosci Bull. 2013 April; 29(2):239-50, Yang et al. Dev Biol. 2011Feb. 1; 350(1):127-38, and Cho et al. Curr Neuropharmacol. 2007 March;5(1): 19-33.

In some embodiments, the compounds can be further screened using an invivo assay that assesses remyelination and reduction of clinicalseverity in the MOG₃₅₋₅₅-induced chronic experimental autoimmuneencephalomyelitis (EAE) rodent model of multiple sclerosis.

Examples of compounds identified by the high-throughput small moleculescreen can include free 1,3-diazoles or free primary diazoles. In someembodiments, the free 1,3-diazoles can have the formula (I):

where R₁ is a substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heteroaryl, heterocycloalkenyl containingfrom 5-6 ring atoms (wherein from 1-3 of the ring atoms is independentlyselected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), O, and S),C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, —Si(C₁-C₃ alkyl)₃, hydroxyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl(—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl),carboxy (—COOH), carboxylato (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano (—CN),isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻), isothiocyanato(—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino(—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido(—NH—(CO)-alkyl), C₆-C₂₀ arylamido (—NH—(CO)-aryl), imino (—CR═NH whereR is hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen, alkyl, aryl,alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl), where R=hydrogen,alkyl, aryl, alkaryl, etc.), nitro (—NO₂), nitroso (—NO), sulfo(—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄ alkylsulfanyl (—S-alkyl; alsotermed “alkylthio”), arylsulfanyl (—S-aryl; also termed “arylthio”),C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl),C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O)₂), phosphinato(—P(O)(O⁻)), phospho (—PO₂), phosphino (—PH₂), or combinations thereof.

In some embodiments, the diazoles can include primary 1,3-diazoles suchas, but not limited to, a primary 1,3-diazole having the formula:

wherein R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are each individually asubstituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄alkynyl, C₃-C₂₀ aryl, heteroaryl, heterocycloalkenyl containing from 5-6ring atoms (wherein from 1-3 of the ring atoms is independently selectedfrom N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), O, and S), C₆-C₂₄alkaryl, C₆-C₂₄ aralkyl, halo, —Si(C₁-C₃ alkyl)₃, hydroxyl, sulfhydryl,C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy,acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, C₂-C₂₄alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato, carbamoyl,C₁-C₂₄ alkyl-carbamoyl, arylcarbamoyl, thiocarbamoyl, carbamido, cyano,isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄alkylamido, C₆-C₂₀ arylamido, imino, alkylimino, arylimino, nitro,nitroso, sulfo, sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄alkylsulfinyl, C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀arylsulfonyl, phosphono, phosphonato, phosphinato, phospho, orphosphino.

In certain specific embodiments, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are eachindividually one or more substituted or unsubstituted groups selectedfrom:

Certain specific embodiments of Formula (I) are shown below:

Compounds described herein may be synthesized using standard synthetictechniques known to those of skill in the art or using methods known inthe an in combination with methods described herein. In additions,solvents, temperatures and other reaction conditions presented hereinmay vary according to the practice and knowledge of those of skill inthe art.

The starting material used for the synthesis of compounds describedherein can be obtained from commercial sources, such as Aldrich ChemicalCo. (Milwaukee, Wis.), Sigma Chemical Co. (St. Louis, Mo.), or thestarting materials can be synthesized. The compounds described herein,and other related compounds having different substituents can besynthesized using techniques and materials known to those of skill inthe art, such as described, for example, in March, ADVANCED ORGANICCHEMISTRY 4^(th) Ed., (Wiley 1992); Carey and Sundberg, ADVANCED ORGANICCHEMISTRY 4^(th) Ed., Vols. A and B (Plenum 2000, 2001), and Green andWuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 3^(rd) Ed., (Wiley 1999)(all of Which are incorporated by reference in their entirety).

In accordance with one aspect of the invention, a method of promotingremyelination in a subject in need thereof is provided. The methodincludes administering to the subject a therapeutically effective amountof at least one free (1,3) diazole compound or free primary diazole,wherein the therapeutically effective amount is the amount effective toinduce endogenous oligodendrocyte precursor differentiation and/ormaturation in the subjects central nervous system.

The oligodendrocyte precursor cell differentiation and/or maturationpromoting compounds can be provided and administered in the form ofpharmaceutical compositions for the in vivo promotion of oligodendrocyteprecursor differentiation and/or maturation. The pharmaceuticalcompositions can be administered to any subject that can experience thebeneficial effects of the oligodendrocyte precursor differentiationand/or maturation compounds of the present invention. Foremost amongsuch animals are humans, although the present invention is not intendedto be so limited.

Pharmaceutical compositions for use in the methods of the presentinvention preferably have a therapeutically effective amount of thecompound or salts thereof in a dosage in the range of 0.01 to 1,000mg/kg of body weight of the subject, and more preferably in the range offrom about 1 to 100 mg/kg of body weight of the patient. In certainembodiments, the pharmaceutical compositions for use in the methods ofthe present invention have a therapeutically effective amount of thecompound or salts thereof in a dosage in the range of 1 to 10 mg/kg ofbody weight of the subject.

The overall dosage will be a therapeutically effective amount dependingon several factors including the particular compound used, overallhealth of a subject, the subject's disease state, severity of thecondition, the observation of improvements, and the formulation androute of administration of the selected agent(s). Determination of atherapeutically effective amount is within the capability of thoseskilled in the art. The exact formulation, route of administration anddosage can be chosen by the individual physician in view of thesubject's condition.

The present invention also provides a method of treating aneurodegenerative disease or disorder in a subject in need thereof byinducing endogenous oligodendrocyte precursor cell (OPC) differentiationand promote myelination in the subject's central nervous system. Themethod includes administering to the subject in need thereof atherapeutically effective amount of a (1,3) Diazole compound inaccordance with the present invention. As described above, one or moreof the compounds can be administered in association with one or morenon-toxic, pharmaceutically acceptable carriers and/or diluents and/oradjuvants and if desired other active ingredients.

The “therapeutically effective amount” of compounds and salts thereofused in the methods of the present invention varies depending upon themanner of administration, the age and body weight of the subject, andthe condition of the subject to be treated, and ultimately will bedecided by those skilled in the art. The term “therapeutically effectiveamount” refers to an amount (dose) effective in treating a subject,having, for example, a neurodegenerative disease (e.g. multiplesclerosis).

In some embodiments, the therapeutically effective amount is the amounteffective to induce endogenous oligodendrocyte precursor cell (OPC)differentiation and/or maturation and thereby promote myelination in thesubject's central nervous system. The induction of endogenousoligodendrocyte precursor cell (OPC) differentiation can becharacterized by a significant increase of myelin basic protein (MBP)expression in newly generated mature myelinating oligodendrocytes in asubject. For example, the increase of MBP expression can be an increaseof MBP expression greater than about 50%, about 100% or about 150% ormore. In certain embodiments, the increase of MBP expression is greaterthan 150% or more compared to a control. In some embodiments, a controlvalue can be readily determined by measuring the MBP expression in asubject's demyleniated lesion prior to administration of apharmaceutical composition described herein.

Differentiated and/or mature oligodendrocytes can be identified by theoligodendrocyte cell body marker CC1 as opposed to the myelinatedprocesses identified using MBP expression. Thus, in some embodiments,the therapeutically effective amount is the amount effective to generateCC1⁺ oligodendrocytes in the subject's central nervous system,particularly in a subject's demyelinated lesion site.

It has been shown that genetic loss of ERK1/2 in the oligodendrocytelineage results in normal numbers of OPCs and oligodendrocytes butwidespread hypomyelination, while constitutive activation of ERK1/2results in a profound increase in the extent of remyelination aftertoxin-induced demyelinating injury. Thus, in some embodiments, thetherapeutically effective amount is the amount effective to induceEKR1/2 phosphorylation and activation in OPCs of the subject.

“Treating” or “treatment” as used herein, refers to the reduction inseverity and/or frequency of symptoms, elimination of symptoms and/orunderlying cause, prevention of the occurrence of symptoms and/or theirunderlying cause, and improvement or remediation of disease. Suchtreatment need not necessarily completely ameliorate the disease. Forexample, treatment of a subject with a neurodegenerative disease byadministration of oligodendrocyte precursor differentiation compounds ofthe present invention can encompass inhibiting or causing regression ofthe disease. Further, such treatment can be used in conjunction withother traditional treatments for neurodegenerative diseases known tothose of skill in the art.

The pharmaceutical compositions of the present invention can beadministered to a subject by any means that achieve their intendedpurpose. For example, administration can be by parenteral, subcutaneous,intravenous, intraarticular, intrathecal, intramuscular,intraperitoneal, or intradermal injections, or by transdermal, buccal,oromucosal, ocular routes or via inhalation. Alternatively, orconcurrently, administration can be by the oral route.

Formulation of the pharmaceutical compounds for use in the modes ofadministration noted above (and others) are known in the art and aredescribed, for example, in Remington's Pharmaceutical Sciences (18thedition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.(also see, e.g., M. J. Rathbone, ed., Oral Mucosal Drug Delivery, Drugsand the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N.Y.,U.S.A., 1996; M. J. Rathbone et al., eds., Modified-Release DrugDelivery Technology, Drugs and the Pharmaceutical Sciences Series,Marcel Dekker, Inc., N.Y., U.S.A., 2003; Ghosh et al., eds., DrugDelivery to the Oral Cavity, Drugs and the Pharmaceutical SciencesSeries, Marcel Dekker, Inc., N.Y., U.S.A., 2005; and Mathiowitz et al.,eds., Bioadhesive Drug Delivery Systems, Drugs and the PharmaceuticalSciences Series, Marcel Dekker, Inc., N.Y., U.S.A., 1999. Compounds ofthe invention can be formulated into pharmaceutical compositionscontaining pharmaceutically acceptable non-toxic excipients andcarriers. The excipients are all components present in thepharmaceutical formulation other than the active ingredient oringredients. Suitable excipients and carriers useful in the presentinvention are composed of materials that are considered safe andeffective and may be administered to an individual without causingundesirable biological side effects, or unwanted interactions with othermedications. Suitable excipients and carriers are those, which arecomposed of materials that will not affect the bioavailability andperformance of the agent. As generally used herein “excipient” includes,but is not limited to surfactants, emulsifiers, emulsion stabilizers,emollients, buffers, solvents, dyes, flavors, binders, fillers,lubricants, and preservatives. Suitable excipients include thosegenerally known in the art such as the “Handbook of PharmaceuticalExcipients”, 4th Ed., Pharmaceutical Press, 2003.

The compounds can be administered to a subject to treatneurodegenerative conditions in subjects in need thereof. Aneurodegenerative disease, as contemplated for treatment by methods ofthe present invention, can arise from but is not limited to stroke, heatstress, head and spinal cord trauma (blunt or infectious pathology), andbleeding that occurs in the brain. Examples of neurodegenerativedisorders contemplated include Alexander disease, Alper's disease,Amyotrophic lateral sclerosis, Ataxia telangiectasia,Spielmeyer-Vogt-Sjogren-Batten disease, Bovine spongiformencephalopathy, Canavan disease, Cockayne syndrome, Corticobasaldegeneration, Creutzfeldt-Jakob disease, Huntington's Disease,HIV-associated dementia, Kennedy's disease, Krabbe disease, Lewy bodydementia, Spinocerebellar ataxias, Multiple Sclerosis, Multiple systematrophy, Neuroborreliosis, Parkinson's disease, Pelizaeus-Merzbacherdisease, Pick's disease, Primary lateral sclerosis, Prion diseases,Refsum's disease, Sandhoff disease, Schilder's disease, Spinal muscularatrophy, Steele-Richardson-Olszewski disease, and tabes dorsalis.

The neurodegenerative disease contemplated for treatment by some aspectsof the present invention can include a myelin related disorder. Myelindisorders can include any disease, condition (e.g., those occurring fromtraumatic spinal cord injury and cerebral infarction), or disorderrelated to demylination, remylination, or dysmyelination in a subject. Amyelin related disorder as used herein can arise from a myelinationrelated disorder or demyelination resulting from a variety of neurotoxicinsults. “Demyelination” as used herein, refers to the act ofdemyelinating, or the loss of the myelin sheath insulating the nerves,and is the hallmark of some neurodegenerative autoimmune diseases,including multiple sclerosis, transverse myelitis, chronic inflammatorydemyelinating polyneuropathy, and Guillain-Barre Syndrome.Leukodystrophies are caused by inherited enzyme deficiencies, whichcause abnormal formation, destruction, and/or abnormal turnover ofmyelin sheaths within the CNS white matter. Both acquired and inheritedmyelin disorders share a poor prognosis leading to major disability.Thus, some embodiments of the present invention can include methods forthe treatment of neurodegenerative autoimmune diseases in a subject. Theterm “remyelination”, as used herein, refers to the re-generation of thenerve's myelin sheath by replacing myelin producing cells or restoringtheir function.

One particular aspect of the present invention contemplates thetreatment of multiple sclerosis in a subject. The method includesadministering to the subject a therapeutically effective amount of oneor more oligodendrocyte differentiation promoting compound(s) describedabove.

Multiple sclerosis (MS) is the most common demyelinating disease. Inmultiple sclerosis, the body's failure to repair myelin is thought tolead to nerve damage, causing multiple sclerosis associated symptoms andincreasing disability. It is contemplated that methods of the presentinvention can promote oligodendrocyte precursor cell differentiation ina subject, therefore leading to endogenous remyelination.

Another strategy for treating a subject suffering from aneurodegenerative disorder such as a myelination related disorder is toadminister a therapeutically effective amount of a free 1,3-diazole orfree primary diazole compound described herein along with atherapeutically effective amount of additional oligodendrocytedifferentiation and/or proliferation inducing agent(s) and/oranti-neurodegenerative disease agent. Therefore, in a further aspect ofthe invention, the 1,3-diazole or free primary diazole oligodendrocyteprecursor differentiation and/or proliferation inducing agents can beadministered as part of a combination therapy with adjunctive therapiesfor treating neurodegenerative and myelin related disorders.

For example, therapeutic methods described herein can further compriseadministering to the subject at least one additional oligodendrocytedifferentiation and/or proliferation inducing agent(s) having theformula (II). Compounds having the formula (II) can be identified by thehigh-throughput small molecule screen described above and can includecompounds having a sterane base such as a steroid hormone or analogthereof. In some embodiments, the sterane base compound can have theformula (II):

where (a) R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇ are eachindividually hydrogen, a substituted or unsubstituted C₁-C₂₄ alkyl,C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heteroaryl,heterocycloalkenyl containing from 5-6 ring atoms (wherein from 1-3 ofthe ring atoms is independently selected from N, NH, N(C₁-C₆ alkyl),NC(O)(C₁-C₆ alkyl), O, and S), C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo,—Si(C₁-C₃ alkyl)₃, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, C₂-C₂₄ alkylcarbonato, C₆-C₂₀arylcarbonato, carboxy, carboxylato, carbamoyl, C₁-C₂₄ alkyl-carbamoyl,arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato,isocyanato, isothiocyanato, azido, formyl, thioformyl, amino, C₁-C₂₄alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido, C₆-C₂₀ arylamido,imino, alkylimino, arylimino, nitro, nitroso, sulfo, sulfonato, C₁-C₂₄alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl, C₅-C₂₀ arylsulfinyl,C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl, phosphono, phosphonato,phosphinato, phospho, or phosphino or combinations thereof, and whereinR₁₀ and R₁₁ may be linked to form a cyclic or polycyclic ring, whereinthe ring is a substituted or unsubstituted aryl, heteroaryl, cycloalkyl,or heterocyclyl;

(b) the ABCD ring structure and/or one or more methyl group areindependently optionally substituted with one or more substituentsselected from C₁-C₆-alkyl, halogenated

C₁-C₆-alkyl, C₁-C₆-alkenyl, halogenated C₁-C₆-alkenyl, halogen, amino,aminoalkylene, hydroxyimino, carbonyl (oxo), and hydroxy;

(c) X is OH or O;

(d) dashed lines are taken at each occurrence independently to be doubleor single bonds; and combinations thereof, or pharmaceuticallyacceptable salts thereof.

In compounds of Formula (II), the ABCD ring structure is the “A”, “B”,“C” and “D” ring portions of a steroid or an analog thereof, which areoptionally substituted; X is O-linked sulfate, OH or O; and wherein thedashed lines can be taken at each occurrence independently to be doubleor single bonds, such to make a valence satisfied and stable molecule.

In some embodiments, optional substituents of the ABCD ring structureinclude one or more of: C₁-C₆-alkyl and halogenated C₁-C₆-alkyl;C₁-C₆-alkenyl and halogenated C₁-C₆-alkenyl, including where the doublebond is directly attached to the ring structure; halogen; amino;aminoalkylene; hydroxyimino; carbonyl (oxo); O-linked sulfate, andhydroxy. Hydrogen substituents on adjacent carbon atoms of the ABCD ringstructure can be optionally removed and replaced by an additional bondbetween the adjacent carbon atoms to result in a double bond betweenthese carbons in the ring structure. In some embodiments, optionalsubstitutions on the ABCD ring structure are methyl groups at one ormore positions of the ring structure.

Certain embodiments of Formula (II) include one or more substituentchosen independently from a hydroxy, or carbonyl (oxo), at any positionof the “A”, “B”, “C” and “D” ring.

Certain specific embodiments of Formula (V) are shown below:

Another example of a compound identified by the high-throughput smallmolecule screen that can be used to promote oligodendrocyte precursordifferentiation and maturation is shown below as Formula (III):

and analogs thereof.

Examples of anti-neurodegenerative disease agents for use in acombination therapy with a 1,3-diazole or free primary diazole caninclude L-dopa, cholinesterase inhibitors, anticholinergics, dopamineagonists, steroids, and immunemodulators including interferons,monoclonal antibodies, and glatiramer acetate.

The phrase “combination therapy” embraces the administration of theendogenous oligodendrocyte precursor differentiation inducing agents anda therapeutic agent as part of a specific treatment regimen intended toprovide a beneficial effect from the co-action of these therapeuticagents. When administered as a combination, the oligodendrocyteprecursor differentiation inducing agents and an additional therapeuticagent can be formulated as separate compositions. Administration ofthese therapeutic agents in combination typically is carried out over adefined time period (usually minutes, hours, days or weeks dependingupon the combination selected).

“Combination therapy” is intended to embrace administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents. Sequential or substantially simultaneousadministration of each therapeutic agent can be effected by anyappropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues. The therapeutic agents can be administered bythe same route or by different routes. For example, a first therapeuticagent of the combination selected may be administered by intravenousinjection while the other therapeutic agents of the combination may beadministered orally. Alternatively, for example, all therapeutic agentsmay be administered orally or all therapeutic agents may be administeredby intravenous injection. The sequence in which the therapeutic agentsare administered is not narrowly critical. “Combination therapy” alsocan embrace the administration of the therapeutic agents as describedabove in further combination with other biologically active ingredients(such as, but not limited to, a second and different therapeutic agent)and non-drug therapies (e.g., surgery).

In another aspect of the invention, the therapeutic agents administeredin a combination therapy with the oligodendrocyte precursor celldifferentiation and/or proliferation inducing agents can include atleast one anti-neurodegenerative agent selected from the groupconsisting of an immunotherapeutic agent.

An immunotherapeutic agent for use in the methods of the presentinvention can include therapies which target the immune component of thedisease and/or the acute inflammatory response evidenced during an acuteattack in remitting-relapsing multiple sclerosis. Examples include, butare not limited to immunomodulators such as interferon beta-la andbeta-lb (Avonex and Betaseron respectively), natalizumab (Copaxone)natalizumab (Tysabri), glatiramer acetate (Copaxone) or mitoxantrone.

The following example is included to demonstrate different embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the example, which follow representtechniques discovered by the inventors to function well in the practiceof the claimed embodiments, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the claims.

Example Drug Based Modulation of Endogenous Stem Cells PromotesFunctional Remyelination In Vivo

As repair of damaged myelin may provide therapeutic benefit in multiplesclerosis (MS) and other demyelinating disorders, we set out to identifydrugs that could be re-purposed as remyelinating therapeutics. Weselected the US National Institutes of Health (NIH) Clinical CollectionI and II libraries comprising 727 drugs with a history of safe use inclinical trials, to test for maturation of oligodendrocyte progenitorcell (OPCs) into myelinating oligodendrocytes. Using mouse epiblast stemcell (EpiSC)-derived OPCs, we developed an in vitro phenotypic screenthat accurately quantified differentiation into mature oligodendrocytesby high content imaging of myelin protein expression (FIG. 1A).

Two batches (>100 million cells each) of pure OPCs were generated fromindependent mouse pluripotent EpiSC lines of opposite sex (FIG. 5A).EpiSC-derived OPCs shared virtually all defining molecular and cellularproperties, including gene expression profiles with in vivo isolatedOPCs, but provided the key advantage of being highly scalable (FIG. 5B).For in vitro screening, the seeding density, endpoint assays, anddimethylsulphoxide (DMSO) (vehicle) tolerance were optimized in pilotstudies to assure accurate and reproducible measurement of OPCdifferentiation in a 96-well format (FIG. 5C).

For the primary screen, OPCs were treated with vehicle alone (0.05%(v/v) DMSO) as a negative control, thyroid hormone (a known OPCdifferentiation inducer) as a positive control, or drug dissolved inDMSO at a concentration of 5 μM. After 72 h, cells were fixed andlabeled with antibodies to myelin basic protein (MBP) and the length andintensity of MBP labeled oligodendrocyte processes measured (FIG. 1A).These features were reliable indicators of alteration in cellularphenotype, as indicated by consistency and high signal to backgroundratio of positive and vehicle controls across all screening plates (FIG.5D-G). We then normalized the experimental data for the tested drugsagainst thyroid hormone (set value of 100) on a per plate basis. On thebasis of this analysis, we identified the 22 drugs that enhancedoligodendrocyte formation greater than five standard deviations aboveDMSO treatment and outperformed thyroid hormone in the measuredparameters (FIG. 1B). Notably, one of the top 22 drugs was benztropine,a muscarinic receptor antagonist recently shown to induce OPCdifferentiation and remyelination.

To validate and prioritize the 22 drug hits, the assay was repeatedusing alternative OPCs, reagents, and parameters to eliminate screenspecific artifacts (see Methods). Drugs were ranked by their dosedependent ability to induce oligodendrocyte generation from OPCs withouttoxicity (FIG. 6A). To demonstrate reproducibility, an independentlaboratory tested selected drug hits using distinct equipment, plateformat (1,536-well), personnel, and imaging/analysis scripts (seeMethods). Of the 16 hits tested at the external screening site, 14 werevalidated as potent inducers of oligodendrocyte differentiation (FIG.6A, B).

We next tested whether the drug hits could promote the maturation ofnative OPCs in central nervous system (CNS) tissue. Cerebellar sliceswere generated from mice at postnatal day 7—a time that precedeswidespread myelination—and treated ex vivo with drug or DMSO (vehicle)for 5 days and labeled with anti-MBP antibodies (FIG. 1C). We screened11 of the top drugs and used a high content analysis (HCA) algorithmdeveloped in house to rank them on the basis of their ability toincrease the extent of MBP1 aligned fibres in whole cerebellar slices.The ‘high’ performing group consisted of four drugs that increased thenumber of MBP aligned fibres ˜150% or greater (FIG. 1D and FIG. 6A). Wevalidated the accuracy of our high content screen by semi-quantitativewestern blotting of MBP protein isoforms in independent slice cultureexperiments (FIG. 1D, E).

Analysis of structure-activity relationships revealed that the top hitsfrom the primary screen segregated into two specific classes containingeither a 1,3-diazole with mono-substitution at the 1-position or asterane base structure (FIG. 7A-D). We selected miconazole andclobetasol, the top overall performing hits in each of the imidazole andsterane classes respectively, for further mechanistic and functionaltesting after confirming that both drugs readily crossed the blood-brainbarrier in mice (FIG. 1F and FIG. 6A). Miconazole is a topicalantifungal agent functioning through cytochrome P450 inhibition, andclobetasol is a potent topical corticosteroid, but their functions inOPCs were unknown.

To test whether miconazole or clobetasol enhance remyelination in vivo,we used a toxin-induced model whereby focal demyelinated lesions aregenerated in dorsal white matter of the spinal cord of adult mice bylocalized injection of lysolecithin (lysophosphatidylcholine (LPC)). Inlesioned animals, demyelination is complete within 4 days, after whichOPCs are recruited into the lesion. Widespread remyelination does notnormally start until 14-21 days post lesion (d.p.l.), which provides adefined window from days 4 to 14 to test the efficacy of drugs toenhance the extent and rate of remyelination. Both miconazole (10 or 40mg per kg (body weight)) and clobetasol (2 mg per kg) treatment induceda marked improvement within the lesions of treated mice compared withvehicle-treated controls. At 8 d.p.l. both drugs induced a significantincrease in the number of newly generated CC1⁺ oligodendrocytes in thelesion core (FIG. 2A, B). This was coincident with extensive MBPstaining in the lesions of miconazole—and clobetasol—but notvehicle-treated animals at both 8 and 12 d.p.l. (FIG. 2A). Electronmicrographs and tissue sections stained with toluidine blue demonstratedthat miconazole and clobetasol each induced a striking increase in theextent of remyelination (FIG. 2C, D and FIG. 8A, B). At 12 d.p.l.,lesions of vehicle-treated mice consisted mostly of unmyelinated axons(6% myelinated) while those of clobetasol- and miconazole-treated micecontained >70% remyelinated axons throughout the extent of the lesion(FIG. 2D). Analysis of myelin thickness relative to axon diameter (gratio) at 12 d.p.l. revealed that miconazole- and clobetasol-inducedmyelin was thinner than intact myelin, a defining characteristic ofremyelination (FIG. 2D).

We also evaluated whether miconazole or clobetasol could promoteprecocious myelination during development, in the absence of injury ordisease. We treated mice at postnatal day 2—a time point that precedeswidespread CNS myelination—daily for 4 days with drug or vehicle. Inmiconazole- and clobetasol-treated mice, we found a significant increasein the number of CC1⁺ oligodendrocytes in the lateral corpus callosumcompared with vehicle-treated mice (FIG. 9A). Additionally, we found asignificantly larger portion of the corpus callosum was populated by MBPfibre tracts in miconazole- and clobetasol-treated mice (Extended DataFIG. 9B). This suggests that clobetasol and miconazole enhancemyelination in the absence of damage or disease. Collectively, the LPCdemyelination and developmental mouse models demonstrate that miconazoleand clobetasol each function to induce the differentiation of endogenousOPCs in the CNS and promote enhanced myelination.

To determine whether the drugs were working at a particular stage of theOPC differentiation process, we seeded OPCs in differentiationconditions and treated them with either miconazole or clobetasol atdifferent time points (0, 16, 24, or 48 h), and assayed MBP expressionat 72 h. For both miconazole and clobetasol, the number of MBPoligodendrocytes present at 72 h was dependent on drug treatment withinthe first 24 h of differentiation (FIG. 3A). In agreement with thesedata, treatment of differentiating OPCs with either drug for differentdurations (24, 48, 56, and 72 h) induced a progressive, time-dependentincrease in the number of MBP oligodendrocytes (FIG. 3B). These datasuggest that both drugs function directly on OPCs early in thedifferentiation process. Additionally, neither drug showed a significantimpact on astrocyte formation from OPCs in vitro, suggesting theyprobably function as direct inducers of oligodendrocyte differentiation(FIG. 3C).

Muscarinic receptor antagonists such as benztropine and clemastine haverecently been identified as remyelinating agents. Therefore we testedwhether miconazole or clobetasol function through the muscarinicacetylcholine pathway using functional cellular reporter assays of allmuscarinic receptor subtypes (M1-M5). Neither miconazole nor clobetasolinhibited any of the five muscarinic receptor subtypes (FIG. 3D). Wethen profiled whether clobetasol or miconazole biochemically inhibitedthe activity of 414 different kinase isoforms. Neither clobetasol normiconazole inhibited any of the kinases tested, suggesting theiractivity is not based on direct inhibition of protein kinases.

To explore the signalling pathways in OPCs influenced by these drugs, weperformed genome-wide RNA sequencing and phosphoproteomic analyses onmouse OPCs treated with drug or vehicle (FIG. 10A-C). Miconazole orclobetasol treatment altered OPC transcript expression andphosphoproteins within hours, and influenced expression of genes insignaling pathways involved in oligodendrocyte maturation andmyelination. Clobetasol potently modulated genes downstream of multiplenuclear hormone receptors, including glucocorticoid receptor, which areknown to be important regulators of myelin gene expression. Sinceglucocorticoid receptor signalling is also known to enhanceSchwann-cell-mediated myelination in the peripheral nervous system, wetested whether the activity of clobetasol on OPCs was mediated byglucocorticoid receptor signalling. Treatment of OPCs with clobetasolfor 1 h increased the phosphorylation of glucocorticoid receptor atSer220, an activating post-translational modification (FIG. 3E). RU486,a competitive glucocorticoid receptor antagonist, blockedclobetasol-induced glucocorticoid receptor phosphorylation andoligodendrocyte differentiation (FIG. 3E, F) suggesting that theactivity of clobetasol in OPCs is mediated through the glucocorticoidreceptor signalling axis.

For miconazole, pathway analyses showed that proteins in themitogen-activated protein (MAP) kinase pathway were most stronglyaffected (FIG. 11A, B). Most Prominent was the strong and sustainedphosphorylation of both extracellular signal-regulated kinases ERK1 andERK2 (ERK1/2) at canonical activation sites, which we validated bywestern blotting (FIG. 3G). In mice, genetic loss of ERK1/2 in theoligodendrocyte lineage results in normal numbers of OPCs andoligodendrocytes but widespread hypomyelination, while constitutiveactivation of ERK1/2 results in a profound increase in the extent ofremyelination after toxin-induced demyelinating injury. In contrasttomiconazole, treatment of OPCs with clobetasol or benztropine did notinduce ERK1/2 phosphorylation (FIG. 3G). Miconazole treatment of anon-neural cell type, mouse fibroblasts, also showed no increase ofERK1/2 phosphorylation, indicating potential cell-type specificity (FIG.3G). PD0325901, a small molecule inhibitor of ERK's upstream MAP-kinasekinase (MEK), blocked the ability of miconazole to induce ERK1/2phosphorylation, suggesting that miconazole functions through aMEK-dependent mechanism in OPCs (FIG. 3H). We also treated mouse OPCswith voriconazole, a triazole-containing antifungal cytochrome P450inhibitor with 80% structural similarity to miconazole, which failed toinduce changes in ERK1/2 phosphorylation (FIG. 3G). This was consistentwith the observation that voriconazole did not promote thedifferentiation of OPCs into oligodendrocytes (FIG. 11C). Takentogether, these results suggest that the effect of miconazole on OPCs isindependent of cytochrome P450 inhibition.

We then assessed whether clobetasol and miconazole treatment wouldenhance the differentiation of human OPCs into oligodendrocytes. Wegenerated human OPCs from human embryonic stem cells (hESCs) andhuman-induced pluripotent stem cells (hiPSCs) (Extended Data FIG. 8A-C).We then treated human OPCs with DMSO, clobetasol, or miconazole for 21days followed by staining for MBP, imaging, and HCA (FIG. 12D-G). Bothdrugs enhanced human OPC differentiation, with miconazole exhibiting themost reproducible and potent effects.

To interpret the potential impact of clobetasol or miconazole astherapeutics in immune-mediated MS models, we tested effects on immunecell survival and function. We found that only clobetasol, as expectedfrom its known corticosteroid properties, altered naïve T-celldifferentiation and both the proliferation and secretion of cytokines byproteolipid protein (PLP₁₃₉₋₁₅₁)- or myelin oligodendrocyte glycoprotein(MOG₃₅₋₅₅)-sensitized lymph node cells (FIG. 13A-J). As such, onlyclobetasol, but not the solely remyelinating drugs miconazole orbenztropine, showed efficacy in reducing disease severity in theimmune-driven relapsing-remitting PLP₁₃₉₋₁₅₁ experimental autoimmuneencephalomyelitis (EAE) model (FIG. 4A). The positive effect ofclobetasol in this model resulted from its immunosuppressive effects asevidenced by the severe reduction of T cells within the spleen (FIG.4B).

We also used a second EAE mouse model, MOG₃₅₋₅₅-induced, in which theimmune response was relatively controlled and disease pathologyrecapitulated chronic progressive demyelination. We used a therapeutic,rather than prophylactic, treatment regimen to evaluate whether drugscould reverse, rather than prevent, disease. Miconazole andclobetasol-treated animals all exhibited a marked improvement infunction, with nearly all animals regaining use of one or both hindlimbs (FIG. 4C, D). In contrast, vehicle-treated mice exhibited chronichindlimb paralysis over the treatment period. Benztropine treatment alsoresulted in functional improvement, but to a lesser extent thanmiconazole and clobetasol (FIG. 4C, D). Overt functional recovery ofmiconazole- and clobetasol-treated mice correlated with histologicalimprovements in the spinal cord. Specifically, drug-treated mice showedrestoration of MBP expression and a reduction in the extent ofdemyelination in the spinal cord, whereas vehicle-treated mice showedsustained areas of white matter disruption (FIG. 14A-E).

Although the immunosuppressive effect of clobetasol makes it challengingto evaluate its remyelinating potential in EAE directly, its consistentand robust induction of OPC differentiation in vitro, and enhancement ofremyelination in non-immune-driven in vivo assays, suggests that itserves a role in both immunomodulation and promotion of myelination. Incontrast, miconazole did not modulate immune cell function and our dataindicate that it acts as a direct remyelinating agent. Given thepotential of miconazole as a remyelinating therapeutic, we contracted aseparate laboratory to provide independent validation of its efficacy inthe MOG₃₅₋₅₅-induced EAE preclinical model. The laboratory independentlyvalidated the preclinical efficacy of miconazole in MOG₃₅₋₅₅-induced EAEto reduce disease severity in treated mice (FIG. 4D).

Since the approval in 1993 of interferon (IFN)-β-1b for the treatment ofMS, therapeutic development has centered on the generation of additionalimmunomodulatory agents. Despite the effectiveness of many of thesedrugs to modulate CNS inflammation in patients with MS, none of themprevent chronic progressive disease and disability—largely because oftheir inability to stop or reverse the failure of remyelination in theCNS. We developed an advanced high throughput screening platform todiscover effective remyelinating therapeutics. This pluripotentstem-cell-based system provides unprecedented scalability, purity, andgenotypic flexibility to screen for compounds that enhance OPCdifferentiation and myelination. Using this platform we identified twodrugs approved by the US Food and Drug Administration, miconazole andclobetasol, with newly discovered functions to modulate OPCdifferentiation directly, enhance remyelination, and significantlyreduce disease severity in mouse models of MS. Miconazole and clobetasolare currently only approved for topical administration in humans.However, the ability of miconazole and clobetasol to cross theblood-brain barrier raises the exciting possibility that these drugs, ormodified derivatives, could advance into clinical trials for thecurrently untreatable chronic progressive phase of MS.

Methods

No statistical methods were used to predetermine sample size.

Mouse OPC Preparation

OPCs used in this study were generated from two separate EpiSC lines,EpiSC9 (female) and 12901 (male), using in vitro differentiationprotocols and culture conditions described previously. Cultures wereregularly tested and shown to be mycoplasma free. To ensure uniformitythroughout all in vitro screening experiments, EpiSC-derived OPCs weresorted to purity by fluorescent activated cell sorting at passage fivewith conjugated CD140a-APC (eBioscience, 17-1401; 1:80) and NG2-AF488(Millipore, AB5320A4; 1:100) antibodies. Sorted batches of OPCs wereexpanded and frozen down in aliquots. OPCs were thawed into growthconditions for one passage before use in screening assays.

In Vitro Phenotypic Screening of OPCs

EpiSC-derived OPCs were seeded onto poly-D-lysine 96-well Viewplate orCellCarrier plates (PerkinElmer) coated with laminin (Sigma, L2020; 10μgml⁻¹) using electronic multichannel pipetors. For the primary screen,30,000 cells were seeded per well in screening medium (DMEM/F12supplemented with N2 (R&D Systems), B-27 (Life Technologies),neurotrophin 3 (R&D Systems; 10 ng ml⁻¹), cAMP (Sigma; 50 μM), IGF-1(R&D Systems; 100 ng ml⁻¹), noggin (R&D Systems; 100 ngml⁻¹)) andallowed to attach for 2 h before addition of drug. NIH ClinicalCollection I and II drugs were added to assay plates with 0.1 ml pinreplicators (Molecular Devices, Genetix; X5051), resulting in a finalprimary screening concentration of 5 μM. Thyroid-hormone-positivecontrols and DMSO vehicle controls were included in each assay plate.Cells were incubated under standard conditions (37° C., 5% CO₂) for 3days and fixed with 4% paraformaldehyde (PFA) in phosphate bufferedsaline (PBS). Fixed plates were permeabilized with 0.1% Triton X-100 andblocked with 10% donkey serum (v/v) in PBS for 2 h. Cells were labelledwith MBP antibodies (Abcam, ab7349; 1:100) for 1 h at room temperature(˜22° C.) followed by detection with Alexa Fluorconjugated secondaryantibodies (1:500) for 45 min. Nuclei were visualized by DAPI staining(Sigma; 1 μgml⁻¹). All plates for the primary screen were processed andanalyzed simultaneously to eliminate variability. Donepezil wasidentified in the primary screen; however, the drug was not available atthe time of dose-response testing and was excluded from further testing.

Dose-response testing of drug hits followed the same procedure with thefollowing modifications to eliminate any artefacts in the primaryscreen: independently sourced drugs; a distinct batch of EpiSC-derivedOPCs from a mouse of opposite sex; multi-dose testing; cytotoxicityanalysis; an alternative marker of mature oligodendrocytes proteolipidprotein 1 (PLP1, antibody clone AA3 provided by B. Trapp; 1:5,000); andan alternative high content assay endpoint parameter (percentage ofoligodendrocytes differentiated instead of process intensity and lengthparameters). All drugs were tested in quadruplicate at seven differentdoses (ranging from 333 nM to 6.7 μM) and classified into tiers on thebasis of their half-maximum effective concentration (EC₅₀) to induce OPCmaturation, and their toxicity (concentration at which 50% of the cellswere lost). Tier A drugs (n=3) consisted of nanomolar dose effectorswith little to no detectable toxicity at doses tested. Tier B drugs(n=4) showed nanomolar effects but demonstrated toxicity at high doses.Tier C and D drugs required high doses to see an effect, demonstratedtoxicity at low doses, or failed to show a dose-dependent response.

HCA of In Vitro Screen

For the 5 μM in vitro screen, stained plates were imaged on the Operaconfocal imaging system (PerkinElmer) and a set of 24×10 fields werecollected from each well, resulting in an average of 10,000 cells beingscored per well. For the dose-response (6.7 μM, 5 μM, 3.3 μM, 1.7 μM,666 nM, 500 nM, and 333 nM) in vitro assays, plates were imaged on theOperetta High Content Imaging and Analysis system (PerkinElmer) and aset of 14×20 fields captured from each well resulting in an average of3,300 cells being scored per well. Analysis (PerkinElmer Acapella,Harmony, and Columbus software) began by identifying intact nucleistained by DAPI; that is, those traced nuclei that were larger than 50μm² in surface area and possessed intensity levels that were typical andless than the threshold brightness of pyknotic cells. Each tracednucleus region was then expanded by 50% and cross-referenced with themature myelin protein (MBP or PLP1) stain to identify oligodendrocytenuclei, and from this the percentage of oligodendrocytes was calculated.Processes emanating from oligodendrocyte nuclei were identified usingthe CSIRO2 analysis module within a custom Acapella script. Maximum meanprocess length (denoted ‘process length’) and mean process intensity(denoted ‘process intensity’) were generated on a per well basis. Forthe 5 μM in vitro screen, values were calculated and normalized to 100for thyroid hormone (positive control)-treated wells and to 0 forDMSO(vehicle)-treated wells, on a per plate basis.

Phenotypic Validation Testing of OPCs

Briefly, OPCs were grown and expanded in laminin-coated flasks beforeharvesting for plating. Cells were dispensed in screening media (seeabove for details) using a Multidrop Combi dispenser (Thermo Fisher)into laminin/poly-L-ornithine-coated sterile, 1,536-well, blackclear-bottom tissue culture plates (Brooks Automation), to a finaldensity of 2,000 cells per well. Plates were sealed with gasketedstainless steel lids with holes for gas exchange (Wako USA). Followingcell attachment, library compounds were transferred by pintool (WakoUSA) using 10 nl slotted pins. Library compounds were serially dilutedin DMSO, and were added to plates to yield final concentrations of 0(DMSO only), 4, and 20 μM compound. After incubation for 72 h at 37° C.,cells were fixed, washed, and stained similar to the 96-well OPC assayprotocol, although all aspiration steps were performed using a BiotekEL406 Microplate Washer Dispenser (Biotek) equipped with a 1,536-wellaspiration manifold. Dispense steps were performed with both peristalticpump cassettes (for gentle reagent additions) and syringe pump manifolds(for faster bulk dispenses). Cells were stained with DAPI (Sigma; 1μgml⁻¹) and MBP antibody (Abcam, ab7349; 1:100). Plates were then imagedusing an InCell 2000 Analyzer High Content Imager (GE HealthcareBio-Sciences). Well images were analysed using InCell AnalyzerWorkstation software, and the MBP signal was quantified with a processdetection algorithm, using total process skeleton length to qualifyactivity.

Ex Vivo Cerebellar Slice Cultures

Whole cerebellum was collected from C57BL/6 mice at postnatal day 7 andembedded in agarose. Sagittal slices were cut on a microtome (Leica) at300 μm. Slices were cultured in a DMEM-Basal Medium Eagle's base with15% heat inactivated horse serum, modified N2, and PDGF-AA. After 1 dayin culture, slices were treated daily for 5 days with test drugs orvehicle (DMSO). Drugs tested were clobetasol (5 μM), hydroxyzine (5 μM),clotrimazole (2 μM), miconazole (1 μM), ketoconazole (1 μM), vesamicol(5 μM), propafenone (2 μM), dicyclomine (5 μM), benztropine (2 μM),haloperidol (5 μM), and medroxyprogesterone (5 μM). The identity of thedrugs was blinded to the experimenter. Slices were then lysed forwestern blot or fixed in 4% PFA and processed for HCA as detailed below.

Immunohistochemistry

Immunohistochemistry was performed as previously described. In short,tissue sections or whole slices were washed three times in PBS, blockedin PBS containing Triton X-100 (0.1%) and normal donkey serum (NDS, 2%for sections and 10% for cerebellar slices) and incubated with primaryantibody overnight. For MBP immunohistochemistry, the primary antibodysolution consisted of 2% NDS, 2% bovine serum albumin, and 0.1% saponin.For all other antibodies, the primary antibody solution consisted of 2%NDS and 0.1% Triton X-100. Primary antibodies used included rat anti-MBP(Abcam, ab7349; 1:100), mouse anti-APC CC1 clone (Millipore, MABC200;1:500), and rabbit anti-IBA1 (Wako Chemicals, 019-19741; 1:1,000). Thetissue was then washed in PBS and incubated in secondary antibodies for2 h. Secondary detection was performed with Alexa Fluor-conjugatedsecondary antibodies (1:500) for 1 h. Luxol fast blue staining wasperformed as previously described.

High Content Screen of Cerebellar Slices

MBP-stained cerebellar slices were analysed by confocal image on anOperetta system using PhenoLOGIC machine-learning technology withinHarmony software. The software was trained to identify elongated fibresmore characteristic of axonal ensheathment and to exclude regions ofsmall fibres or diffuse background fluorescence on the basis of texturefeatures. MBP-positive surface area was collected and normalized to thetotal surface area for the group of slices treated with each drug. Aminimum of six slices were treated per drug, which included an equaldistribution of medial and lateral slices.

Western Blotting of Cerebellar Slices

Cerebellar slices (each biological replicate using 12 slices percondition; six each from two separate animals) were collected in PBS andcentrifuged. The PBS was aspirated and the pellet resuspended in 100 mLlysis buffer (20 mM Tris, 137 mM NaCl, 5.0 mM EDTA pH 8.0, 10% glycerol,1% NP40, pH to 8.0 with HCl), incubated on ice for 20 min, centrifuged,and the supernatant collected. Protein concentration was determined by aPierce BCA protein assay kit (Thermo Fisher). Equal amounts of proteinwere applied to NuPAGE 12% Bis-TRIS gels (Life Technologies), andelectrophoretically transferred onto a PVDF membrane (LifeTechnologies). The membranes were incubated with rabbit anti-MBP(Millipore, AB980; 1:500) and consequently probed with horseradishperoxidase (HRP)-conjugated goat anti-rabbit (1:5,000) or incubated withHRP-conjugated mouse anti-β-actin (Sigma, A3854; 1:10,000) to ensureeven loading of samples. Enhanced chemiluminescence was performed with aWest Pico kit (Thermo Fisher) and relative optical density was measuredusing ImageJ (NIH).

Chemoinformatics

Structure-activity searches of azoles and steranes were performed withCanvas program (Schrodinger Software, release 2014-1: Canvas, version1.9). Tanimoto similarity between voriconazole and miconazole wascalculated by ROCS (OpenEye Scientific Software).

Pharmacokinetics

C57BL/6 adult female mice were dosed intraperitoneally with miconazole(10 mg/kg or 40 mg/kg) or clobetasol (10 mg/kg). After 1 or 6 h, 100 mlof plasma was collected then each animal was perfused with PBS. Brainswere collected, weighed, and rinsed with PBS. Water (0.5 ml) was addedto the brain samples, which were then homogenized. Plasma and brainsamples were each diluted fivefold with blank rat plasma. Three hundredmicroliters of internal standard solution was added to the samples,vortexed, and centrifuged. Five microliters of each sample was injectedinto an API-4000Qtrap mass spectrometer and quantified (Climax Labs).

Focal Demyelination and Drug Treatment

Focal demyelination in the spinal cord was induced by the injection of1% LPC solution. Ten- to 12-week-old C57BL/6 female mice wereanaesthetized using isoflurane and a T10 laminectomy was performed. Onemicroliter of 1% LPC was infused into the dorsal column at a rate of 15ml h⁻¹. The animals were euthanized either at day 8 or day 12 after thelaminectomy (n=6-9 per group). Animals that were euthanized at day 8received vehicle or drug daily by intraperitoneal injection between days3 and 7. Animals used in the day 12 experiments received vehicle or drugdaily by intraperitoneal injection between days 4 and 11. Drugs weredissolved in DMSO and then diluted with sterile saline for injection.Mice were deeply anaesthetized using ketamine/xylazine rodent cocktailand then euthanized by transcardial perfusion with 4% PFA forhistological analysis or 4% PFA, 2% gluteraldehyde, and 0.1M sodiumcacodylate for electron microscopy. PFA fixed tissue was equilibrated in30% sucrose, embedded in OCT, and cryosectioned at 20 μm thickness andprocessed for CC1 and MBP immunohistochemistry. ImageJ was used tomeasure area of the lesion and CC1⁺ cells within the lesion were scoredmanually. For CC1 scoring, sections were taken from the centre of eachlesion to control for lesion variability.

Electron Microscopy

Samples were processed as previously described. In short, samples wereosmicated, stained en bloc with uranyl acetate, and embedded in EMbed812, an Epon-812 substitute (EMS). Sections (1 μm) were cut and stainedwith toluidine blue and visualized on a light microscope (LeicaDM5500B). Additional thin sections were cut, carbon coated and imagedeither on a JEOL JEM-1200-EX electron microscope or a T12 electronmicroscope (FEI).

Developmental Myelination

Mouse pups of strain CD1 were administered 2 mg/kg clobetasol, 10 mg/kgmiconazole, or vehicle (DMSO in saline) by daily intraperitonealinjections from postnatal day 2-6. Some clobetasol treated animalsexhibited sickness on the basis of this treatment, with low body weight,and some animals of the cohort died before end of treatment. Onpostnatal day 6 the pups were anaesthetized using ketamine and xylazineand euthanized by transcardial perfusion with 4% PFA. Tissue was fixedovernight in 4% PFA, equilibrated in 30% sucrose, and embedded in OCT.Sections (20 μm) were cut and processed for CC1 and MBPimmunohistochemistry. ImageJ was then used to count and measure area ofthe corpus callosum as well as measure the extent of the corpus callosumlength covered by MBP1 processes. Eight coronal sections containingcorpus callosum rostral to the hippocampus from at least three animalsper group were used for these analyses. To quantitate extent of MBP inthe corpus callosum, a line was drawn through the centre of the corpuscallosum from the lateral tip to the dorsal most extent of MBPexpression in the corpus callosum. The length of this line was measuredand then the dorsal-most point of the line was extended to the dorsaltip of the corpus callosum and measured to yield the length of thelateral callosum. The two numbers were divided to get the MBP/corpuscallosum proportion. A two-tailed t-test was used to compare drug-withvehicle-treated groups.

Muscarinic Receptor Antagonism

Miconazole, clobetasol, and benztropine (all at 1 μM in DMSO) were sentto Select Screen (Life Technologies) with identities coded. GeneBLAzeror Tango assays were performed to determine level of acetylcholinemuscarinic receptor M1, M3, M5 (GeneBLAzer), M2 and M5 (Tango)antagonism.

Kinase Profiling

LanthaScreen, Z9-LYTE, and Adapta kinase assays were performed by SelectScreen (Life Technologies). LanthaScreen Eu kinase assays were performedin Greiner low-volume 384-well plates. Assay buffer consisted of 50 mMHEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl₂, 1 mM EGTA. Each well consistedof this mixture: 4.0 μl of 4 μM test drug in assay buffer, 8 μl of 2×kinase/Eu antibody mixture, and 4 μl of 4× Alexa Fluor 647 tracer.Plates were incubated for 60 min at room temperature (˜22° C.), thenAlexa Fluor 647 emission (665 nm) and Europium emission (615 nm) read ona fluorescent plate reader. Data were analysed by generating theemission ratio (665 nm/615 nm) for each test point and normalizing 0% tocontrol wells with no known inhibitor and 100% to control wells withhighest concentration of known inhibitor.

Z′-LYTE assays were performed in Corning, low volume 384-well plates.Assay buffer consisted of 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mMMgCl₂, 1 mM EGTA. Each well consisted of this mixture: 2.5 μl 4× of 4 μMdrug in assay buffer, 5 μl 2× peptide/kinase mixture, 2.5 μl 4×ATPsolution. Plates were then incubated at room temperature (˜22° C.) for60 min. Then 5 μl of a development reagent that contained a proteasethat selectively digested the non-phosphorylated peptide was added andthe plates incubated for 60 min. Coumarin emission (445 nm) andfluorescein emission (520 nm) were read on a fluorescent plate reader.Data were analysed by normalizing out background fluorescence thengenerating the emission ratio (445 nm/520 nm) for each test point. Datawere further normalized to 0% in control wells with no ATP and 100% incontrol wells with synthetically phosphorylated peptide of the samesequence.

Adapta assays were performed in Corning, low volume 384-well plates.Assay buffer consisted of 30 mM HEPES. Each well consisted of thismixture: 2.5 μl 4× of 4 μM drug in assay buffer, 2.5 μl 4×ATP solution,5 μl 2× substrate/kinase mixture. Plates were then incubated at roomtemperature (˜22° C.) for 60 min. Then 5 μl of a development reagentthat contained europium-anti-ADP antibody and ADP tracer were added andthe plate incubated for 60 min. Alexa Fluor 647 emission (665 nm) andeuropium emission (615 nm) were read on a fluorescent plate reader. Datawere analysed by generating the emission ratio (665 nm/615 nm) for eachtest point and normalizing 0% to control wells with no ATP in the kinasereaction and 100% to control wells with ADP.

HCA of Astrocyte Induction

For the astrocyte experiments in FIG. 3C, the experimental setup wasidentical to the PLP1-based primary validation screen except plates werestained for GFAP (DAKO, Z0334; 1:5,000). BMP4 (R&D Systems; 50 ng ml⁻¹)and LIF (Millipore; 10³ U ml⁻¹) were used as the positive control forastrocyte induction. Assay plates were imaged on the Operetta HighContent Imaging and Analysis system and a set of 14×20 fields captured.Columbus Data Management and Analysis System software (PerkinElmer) wasused to quantify the percentage of GFAP1 astrocytes in each well using amethod similar to that developed for oligodendrocytes.

Global Phosphoproteomics

Quantitative global phosphorylation studies were performed on OPCsacross two different time points (1 and 5 h after treatment) withmiconazole, clobetasol, or DMSO treatment using a label-free ultra-highperformance liquid chromatography tandem mass spectrometry (LC-MS/MS)workflow without fractionation. Briefly, for each sample 30 millioncells were lysed with 2% SDS solution with protease and phosphataseinhibitor (Thermo Fisher), and detergent was removed on 200 μl of thecell lysate using the FASP cleaning procedure. Each sample was digestedby a two-step Lys-C/trypsin proteolytic cleavage and subjected tophospho-enrichment using a commercially available TiO2 enrichment spintips (Thermo Fisher). LC-MS/MS analysis used a UPLC system (NanoAcquity,Waters) that was interfaced to an Orbitrap ProVelos Elite MS system(Thermo Fisher). Fold-change calculations were determined from peptideintensities for each drug versus DMSO at each time point.Phosphopeptides with greater than twofold change were imported intoIngenuity Pathway Analysis to elucidate signalling pathways perturbedwith drug treatment.

RNA Sequencing and Analysis

Cells were lysed directly in 1 ml TRIzol (Invitrogen) and stored at −80°C. Once all samples were collected, samples were thawed on ice andseparated with chloroform using Phase Lock Gel tubes (5 PRIME). RNA wasisolated using the miRNeasy PlusMini Kit (Qiagen) according to themanufacturer's protocol. One microgram of each sample was then poly-Aselected, fragmented, and library prepared using the TruSeqRNA SamplePrep Kit (Illumina) according to the manufacturer's protocol. Sampleswere indexed using TruSeq adapters. One hundred base-pair paired-endreads were generated for each sample on an Illumina HiSeq 2500instrument at the Case Western Reserve University Genomics Corefacility. Between 5 million and 13 million reads were generated persample for drug time course experiments. EpiSC RNaseq data werepreviously published (GEOaccession number GSE57403). EpiSCs, EpiSC OPCs,and in vivo OPCs were sequenced to depths of 51,271,458 reads,61,072,460 reads, and 62,530,709 reads, respectively. For in vivoisolated OPCs, CD140a⁺ cells were immunopanned from the CNS of mousepups at postnatal day 7 as described previously. Cells were thencultured for 5 days in identical culture conditions to EpiSC-derivedOPCs before analysis.

Reference genome files were retrieved from Illumina iGenomes. Reads werealigned to the mm9 genome using Tophat version 2.0.8 with defaultsettings. Expression values of known RefSeq genes were calculated inunits of fragments per kilobase per million reads (FPKM) using Cufflinksversion 2.0.2. Expression values were tabled to eliminate backgroundsignal by converting all values below 0.25 to 0, and subsequently adding0.25 to all values. FPKMs were quantile normalized to correct forinter-sample variation. To identify genes whose expression was perturbedby drug treatments, duplicate samples of OPCs were treated with drug orvehicle for 2, 6, or 12 h. RNA sequencing data were tested fordifferential expression by comparing treatments to vehicle at each timepoint using Cuffdiff version 2.0.2. The collective list of changed genesfor each drug was evaluated with Ingenuity Pathway Analysis (applicationbuild 261899, content version 18030641).

Western Blotting of Mouse OPCs

EpiSC-derived OPCs were seeded into poly-Lornithine/laminin coatedsix-well plates and allowed to attach for 2 h in DMEM/F12 withoutadditional factors. Cells were treated with indicated inhibitors or DMSOfor 1 h—SCH772984 (ChemieTek, 1 mM), SB590885 (Tocris, 10 nM), LY294002(Tocris, 10 μM), and PD0325901 (Stemgent, 1 μM). Cells were thenstimulated with drug or FGF2 (R&D Systems; 20 ng ml⁻¹) for 1 h and thenlysed in 200 μL RIPA buffer (0.15 M NaCl, 0.05M Tris, pH=8.0, 1 mM EDTA,1% Triton X-100, 0.1% SDS, 10% glycerol, HALT protease and phosphataseinhibitor (Thermo Fisher) added just before use) and incubated on icefor 20 min. Lysates were centrifuged at 4° C. and supernatant collected.Protein concentrations were determined by Pierce BCA protein assay kit(Thermo Fisher). Equal amounts of protein were resolved in a reducedmanner on NuPAGE 4-12% Bis-Tris gels (Life Technologies) and transferredonto PVDF membranes (Life Technologies). Blots were blocked in either 5%BSA (phosphoprotein) or 5% milk (non phosphoprotein). Primary antibodieswere all from the same vendor (Cell Signaling) and includedphospho-Erk1/2 (4370S, clone D13.14.4E; 1:2,000), ERK1/2 (9107S, clone3A7; 1:2,000), phospho-glucocorticoid receptor (4161S; 1:1,000), andglucocorticoid receptor (12041, clone D6H2L; 1:1,000) followed byincubation with HRP-conjugated secondary antibodies and chemiluminescentenhancement by West Pico substrate (Thermo Fisher).

Generation and Screening of Human OPCs

Human OPCs were generated from skin fibroblast-derived human iPSC line(CWRU43, Tesar laboratory) and hESC lines H7 (NIH Human EmbryonicStemCell RegistryWA07; NIH approval number NIHhESC-10-0061) and H9 (NIHHuman Embryonic Stem Cell Registry WA09; NIH approval numberNIHhESC-10-0062) as previously described. iPSC- and hESC-derived OPCswere characterized by Sox10 (R&D Systems, AF2864; 1:100) staining, andthen seeded in 96-well plates at 40,000 cells per well for drug testing.Cells were cultured with 1 μM miconazole, 5 μM clobetasol, or vehicle(DMSO) for 21 days, with fresh media changes with drug or vehicle every2 days. Plates were fixed and stained with MBP (Abcam, ab7349; 1:100)then imaged on the Operetta system. We analysed results with slightmodification to HCA Acapella scripts used for mouse oligodendrocytes.

Naive CD41 T-Cell Assays

Naive CD4+ T cells (CD4+L-selectin^(hi) cells) were purified usingAutoMacs Magnetic Bead cell separation technology (Miltenyi Biotech)from total lymph node cells isolated from unprimed mice with purityranging from 98 to 99.9%. For in vitro activation, 5×10⁵ naive CD4⁺ Tcells were activated in the presence of plate-bound anti CD3 (1 μgml⁻¹)plus Th1- (200 U ml⁻¹ interleukin-2 (IL-2), 40 U ml⁻¹ IL-12, 10 μgml⁻¹anti-IL-4) or Th17- (10 μg ml⁻¹ TGF-β1, 50 ng ml⁻¹ IL-6, 1 μgml⁻¹anti-IFN-γ, 1 μgml⁻¹ anti-IL-4, 1 μgml⁻¹ anti-IL-2) promotingconditions. On day 4, the cultured T cells were collected and thepercentage of viable cytokine positive cells assessed by flow cytometry.The cells were stained with a LIVE/DEAD Fixable Violet Dead Cell StainKit, for 405 nm excitation (Life Technologies), anti CD4-APC/Cy7 (cloneRM4-5), anti-IFN-γ-PerCP/Cy5.5 (clone XMG1.2), and anti-IL-17-APC (cloneeBio17B7) (eBioscience). Viable cells (5×10⁵) were analysed perindividual sample using a BD Canto II cytometer (BD Biosciences), andthe data were analysed using FloJo version 9.5.2 software (Tree Star).

Ex Vivo Lymphocyte Recall Assays

Female SJL/J (Harlan Laboratories) or C57BL/6 mice were housed under SPFconditions. Six- to seven-week-old female mice were immunizedsubcutaneously with 100 μl of an emulsion containing 200 μg ofMycobacterium tuberculosis H37Ra (BD Biosciences) and 50 μg ofPLP₁₃₉₋₁₅₁ (SJL/J) or MOG₃₅₋₅₅ (C57BL/6) distributed over three sites onthe flank. For ex vivo culture draining, lymph nodes on day 8 werecollected and cells were activated in the presence of anti-CD3 (1μgml⁻¹) in the absence or presence of clobetasol, miconazole, orbenztropine (10⁻⁹-10⁻⁵ M). To assess total cellular proliferation,cultures were pulsed with tritiated thymidine (1 μCi) at 24 h andcultures were harvested at 72 h. In replicate wells, culturesupernatants were harvested at 72 h after culture, and the level of IFNγ and IL-17 were assessed via Luminex assay (Millipore).

PLP₁₃₉₋₁₅₁-Induced Relapsing Remitting EAE

Six- to seven-week-old female SJL/J mice were induced with PLP₁₃₉₋₁₅₁ asfor ex vivo recall assays. Mice were allowed to progress to diseaseonset at day 13 before being randomized into treatment groups (n=10 miceper group). Mice were then monitored for paralysis and treated daily byintraperitoneal injection with vehicle (DMSO in sterile saline),benztropine (10 mg/kg), clobetasol (2 mg/kg), or miconazole (10 mg/kg)beginning on day 13 and ending on day 29. This period fully encompassedthe acute phase of disease onset followed by remission and the primarydisease relapse. Treatments were blinded to the experimenters performingthe assays. Mice were followed for disease severity in a blinded fashionwith disease scoring as follows: 0, no abnormality; 1, limp tail; 2,limp tail and hind limb weakness; 3, hind limb paralysis; 4, hind limbparalysis and forelimb weakness; and 5, moribund.

MOG₃₅₋₅₅ Chronic Progressive EAE Model

EAE was induced by immunizing 10-week-old C57BL/6 female mice with 100ml injection of MOG₃₅₋₅₅/complete Freund's adjuvant emulsion (HookeLaboratories). One hour after immunization, mice were given 100 ng ofpertussis toxin by 100 μl intraperitoneal injection. A second dose ofpertussis toxin was administered the next day. EAE onset was monitoreddaily and scored using a clinical scale where 0 represented noabnormality; 1, limp tail; 2, limp tail and hind limb weakness; 3, hindlimb paralysis; 4, hind limb paralysis and forelimb weakness; and 5,moribund. Mice that appeared moribund or exhibited forelimb paralysiswere immediately euthanized and not used for the study. Once micereached peak of disease ˜day 15; clinical score=3) they were randomizedinto treatment groups, and drug or vehicle (DMSO in saline) wasadministered intraperitoneally daily for 10 days (n=12-16 mice pergroup). Doses for each drug were miconazole (10 mg/kg), clobetasol (2mg/kg), and benztropine (10 mg/kg). At these doses, no drugs showed anyovert evidence of toxicity to any of the animals. Experimenters wereblinded to the identity of the treatments and animals were scored daily.Cumulative disease scores for each animal were calculated during thetreatment period, and a two-tailed t-test compared drug-withvehicle-treated groups. The extent of recovery for each animal wascalculated as the difference between the peak disease score and thescore at the end of each experiment, and a two-tailed t-test was used tocompare each treatment with vehicle. External validation of MOG₃₅₋₅₅ EAEexperiments (n=12 mice per group) was performed at Hooke Laboratorieswith experimenters blinded to the identity of the substances. FTY720(fingolimod, 1 mg/kg), a known immunomodulatory drug, was used as apositive control during external validation of miconazole (10 mg/kg).

While this invention has been shown and described with references tovarious embodiments thereof, it will be understood by those skilled inthe art that changes in form and details may be made therein withoutdeparting from the scope of the invention encompassed by the appendedclaims. All patents, publications and references cited in the foregoingspecification are herein incorporated by reference in their entirety.

What is claimed is:
 1. A method of promoting remyelination in a subjectin need thereof the method comprising administering to the subject atherapeutically effective amount of at least one (1,3) Diazole compound,wherein the therapeutically effective amount is the amount effective toinduce endogenous oligodendrocyte precursor cell (OPC) differentiationin the subject's central nervous system.
 2. The method of claim 1, theat least one (1,3) diazole compound or analog thereof having the formula(I):

where R₁ is a substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heteroaryl, heterocycloalkenyl containingfrom 5-6 ring atoms (wherein from 1-3 of the ring atoms is independentlyselected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), O, and S),C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, —Si(C₁-C₃ alkyl)₃, hydroxyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl,C₂-C₂₄ alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato,carbamoyl, C₁-C₂₄ alkyl-carbamoyl, arylcarbamoyl, thiocarbamoyl,carbamido, cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido,formyl, thioformyl, amino, C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄alkylamido, C₆-C₂₀ arylamido, imino, alkylimino, arylimino, nitro,nitroso, sulfo, sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄alkylsulfinyl, C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀arylsulfonyl, phosphono, phosphonato, phosphinato, phospho, phosphino,and combinations thereof, or pharmaceutically acceptable salts thereof.3. The method of claim 1, the at least one compound or analog thereofcomprising a primary 1,3-diazole.
 4. The method of claim 3, the primary1,3-diazole having the formula:

wherein R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are each individually asubstituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄alkynyl, C₃-C₂₀ aryl, heteroaryl, heterocycloalkenyl containing from 5-6ring atoms (wherein from 1-3 of the ring atoms is independently selectedfrom N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), O, and S), C₆-C₂₄alkaryl, C₆-C₂₄ aralkyl, halo, —Si(C₁-C₃ alkyl)₃, hydroxyl, sulfhydryl,C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy,acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, C₂-C₂₄alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato, carbamoyl,C₁-C₂₄ alkyl-carbamoyl, arylcarbamoyl, thiocarbamoyl, carbamido, cyano,isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄alkylamido, C₆-C₂₀ arylamido, imino, alkylimino, arylimino, nitro,nitroso, sulfo, sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄alkylsulfinyl, C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀arylsulfonyl, phosphono, phosphonato, phosphinato, phospho, orphosphino.
 5. The method of claim 4, wherein R₂, R₃, R₄, R₅, R₆, R₇, andR₈ are each individually one or more substituted or unsubstituted groupsselected from:


6. The method of claim 1, the at least one compound having the formula:

or analog thereof.
 7. The method of claim 1, wherein the induction ofendogenous oligodendrocyte precursor cell (OPC) differentiation ischaracterized by an increase of myelin basic protein (MBP) expression.8. The method of claim 7, wherein the increase of MBP expression isgreater than 150% or more compared to a control.
 9. The method of claim1, wherein the therapeutically effective amount is an amount effectiveto induce EKR1/2 phosphorylation in OPCs of the subject.
 10. The methodof claim 1, wherein the therapeutically effective amount is an amounteffective to generate CC1+ oligodendrocytes in the subjects centralnervous system.
 11. The method of claim 1, the subject in need having orsuspected of having a myelin related disorder.
 12. The method of claim1, wherein remyelination is promoted in a subject's CNS demyelinatedlesion related to the myelin related disorder.
 13. The method of claim11, the myelination related disorder selected from multiple sclerosis,transverse myelitis, chronic inflammatory demyelinating polyneuropathy,and Guillain-Barre Syndrome.
 14. The method of claim 11, wherein themyelin related disorder is multiple sclerosis.
 15. The method of claim1, wherein the at least one (1,3) diazole compound is administeredsystemically.
 16. The method of claim 1, further comprisingadministering to the subject at least one compound having the formula(II):

where (a) R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇ are eachindividually hydrogen, a substituted or unsubstituted C₁-C₂₄ alkyl,C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heteroaryl,heterocycloalkenyl containing from 5-6 ring atoms (wherein from 1-3 ofthe ring atoms is independently selected from N, NH, N(C₁-C₆ alkyl),NC(O)(C₁-C₆ alkyl), O, and S), C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo,—Si(C₁-C₃ alkyl)₃, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, C₂-C₂₄ alkylcarbonato, C₆-C₂₀arylcarbonato, carboxy, carboxylato, carbamoyl, C₁-C₂₄ alkyl-carbamoyl,arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato,isocyanato, isothiocyanato, azido, formyl, thioformyl, amino, C₁-C₂₄alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido, C₆-C₂₀ arylamido,imino, alkylimino, arylimino, nitro, nitroso, sulfo, sulfonato, C₁-C₂₄alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl, C₅-C₂₀ arylsulfinyl,C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl, phosphono, phosphonato,phosphinato, phospho, or phosphino or combinations thereof, and whereinR₁₀ and R₁₁ may be linked to form a cyclic or polycyclic ring, whereinthe ring is a substituted or unsubstituted aryl, heteroaryl, cycloalkyl,or heterocyclyl; (b) the ABCD ring structure and/or one or more methylgroup are independently optionally substituted with one or moresubstituents selected from C₁-C₆-alkyl, halogenated C₁-C₆-alkyl,C₁-C₆-alkenyl, halogenated C₁-C₆-alkenyl, halogen, amino, aminoalkylene,hydroxyimino, carbonyl (oxo), and hydroxy; (c) X is OH or O; (d) dashedlines are taken at each occurrence independently to be double or singlebonds; and combinations thereof, or pharmaceutically acceptable saltsthereof.
 17. The method of claim 16, wherein R₉ is

CH₃, OCH₃ or an alkyl halide.
 18. The method of claim 16, wherein R₁₀ isH, hydroxyl or


19. The method of claim 16, the at least one compound or analog thereofhaving the formula:

or an analog thereof.
 20. A method of treating a neurodegenerativedisease or disorder in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of atleast one (1,3) Diazole compound, wherein the therapeutically effectiveamount is the amount effective to induce endogenous oligodendrocyteprecursor cell (OPC) differentiation and promote myelination in thesubject's central nervous system.
 21. The method of claim 20, the atleast one (1,3) diazole compound or analog thereof having the formula(I):

where R₁ is a substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heteroaryl, heterocycloalkenyl containingfrom 5-6 ring atoms (wherein from 1-3 of the ring atoms is independentlyselected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), O, and S),C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, —Si(C₁-C₃ alkyl)₃, hydroxyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl,C₂-C₂₄ alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato,carbamoyl, C₁-C₂₄ alkyl-carbamoyl, arylcarbamoyl, thiocarbamoyl,carbamido, cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido,formyl, thioformyl, amino, C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄alkylamido, C₆-C₂₀ arylamido, imino, alkylimino, arylimino, nitro,nitroso, sulfo, sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄alkylsulfinyl, C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀arylsulfonyl, phosphono, phosphonato, phosphinato, phospho, phosphino,and combinations thereof, or pharmaceutically acceptable salts thereof.22. The method of claim 21, the at least one compound or analog thereofcomprising a primary 1,3-diazole.
 23. The method of claim 22, theprimary 1,3-diazole having the formula:

wherein R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are each individually asubstituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄alkynyl, C₃-C₂₀ aryl, heteroaryl, heterocycloalkenyl containing from 5-6ring atoms (wherein from 1-3 of the ring atoms is independently selectedfrom N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), O, and S), C₆-C₂₄alkaryl, C₆-C₂₄ aralkyl, halo, —Si(C₁-C₃ alkyl)₃, hydroxyl, sulfhydryl,C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy,acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, C₂-C₂₄alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato, carbamoyl,C₁-C₂₄ alkyl-carbamoyl, arylcarbamoyl, thiocarbamoyl, carbamido, cyano,isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄alkylamido, C₆-C₂₀ arylamido, imino, alkylimino, arylimino, nitro,nitroso, sulfo, sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄alkylsulfinyl, C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀arylsulfonyl, phosphono, phosphonato, phosphinato, phospho, orphosphino.
 24. The method of claim 23, wherein R₂, R₃, R₄, R₅, R₆, R₇,and R₈ are each individually one or more substituted or unsubstitutedgroups selected from:


25. The method of claim 20, the at least one compound having theformula:

or analog thereof.
 26. The method of claim 20, wherein the induction ofendogenous oligodendrocyte precursor cell (OPC) differentiation ischaracterized by an increase of myelin basic protein (MBP) expression.27. The method of claim 26, wherein the increase of MBP expression isgreater than 150% or more compared to a control.
 28. The method of claim20, wherein the therapeutically effective amount is an amount effectiveto induce EKR1/2 phosphorylation in OPCs of the subject.
 29. The methodof claim 20, wherein the therapeutically effective amount is an amounteffective to generate CC1+ oligodendrocytes in the subject's centralnervous system.
 30. The method of claim 20, the neurodegenerativedisease or disorder comprising a myelin related disorder.
 31. The methodof claim 30, the myelination related disorder selected from multiplesclerosis, transverse myelitis, chronic inflammatory demyelinatingpolyneuropathy, and Guillain-Barre Syndrome.
 32. The method of claim 30,wherein the myelin related disorder is multiple sclerosis.
 33. Themethod of claim 20, wherein the at least one (1,3) diazole compound isadministered systemically.
 34. The method of claim 20, furthercomprising administering to the subject at least one compound having theformula (II):

where (a) R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇ are eachindividually hydrogen, a substituted or unsubstituted C₁-C₂₄ alkyl,C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heteroaryl,heterocycloalkenyl containing from 5-6 ring atoms (wherein from 1-3 ofthe ring atoms is independently selected from N, NH, N(C₁-C₆ alkyl),NC(O)(C₁-C₆ alkyl), O, and S), C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo,—Si(C₁-C₃ alkyl)₃, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, C₂-C₂₄ alkylcarbonato, C₆-C₂₀arylcarbonato, carboxy, carboxylato, carbamoyl, C₁-C₂₄ alkyl-carbamoyl,arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato,isocyanato, isothiocyanato, azido, formyl, thioformyl, amino, C₁-C₂₄alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido, C₆-C₂₀ arylamido,imino, alkylimino, arylimino, nitro, nitroso, sulfo, sulfonato, C₁-C₂₄alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl, C₅-C₂₀ arylsulfinyl,C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl, phosphono, phosphonato,phosphinato, phospho, or phosphino or combinations thereof, and whereinR₁₀ and R₁₁ may be linked to form a cyclic or polycyclic ring, whereinthe ring is a substituted or unsubstituted aryl, heteroaryl, cycloalkyl,or heterocyclyl; (b) the ABCD ring structure and/or one or more methylgroup are independently optionally substituted with one or moresubstituents selected from C₁-C₆-alkyl, halogenated C₁-C₆-alkyl,C₁-C₆-alkenyl, halogenated C₁-C₆-alkenyl, halogen, amino, aminoalkylene,hydroxyimino, carbonyl (oxo), and hydroxy; (c) X is OH or O; (d) dashedlines are taken at each occurrence independently to be double or singlebonds; and combinations thereof, or pharmaceutically acceptable saltsthereof.
 35. The method of claim 34, wherein R₉ is

CH₃, OCH₃ or an alkyl halide.
 36. The method of claim 34, wherein R₁₀ isH, hydroxyl or


37. The method of claim 34, the at least one compound or analog thereofhaving the formula:

or an analog thereof.
 38. A method of treating a myelin related diseasein a subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of at least one (1,3) Diazolecompound, the at least one (1,3) diazole compound or analog thereofhaving the formula (I):

where R₁ is a substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heteroaryl, heterocycloalkenyl containingfrom 5-6 ring atoms (wherein from 1-3 of the ring atoms is independentlyselected from N, NH, N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), O, and S),C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, —Si(C₁-C₃ alkyl)₃, hydroxyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl,C₂-C₂₄ alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato,carbamoyl, C₁-C₂₄ alkyl-carbamoyl, arylcarbamoyl, thiocarbamoyl,carbamido, cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido,formyl, thioformyl, amino, C₁-C₂₄ alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄alkylamido, C₆-C₂₀ arylamido, imino, alkylimino, arylimino, nitro,nitroso, sulfo, sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄alkylsulfinyl, C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀arylsulfonyl, phosphono, phosphonato, phosphinato, phospho, phosphino,and combinations thereof, or pharmaceutically acceptable salts thereof,wherein the therapeutically effective amount is the amount effective toinduce endogenous oligodendrocyte precursor cell (OPC) differentiationand promote myelination in the subject's central nervous system.
 39. Themethod of claim 38, wherein the myelin related disorder is multiplesclerosis.